This study presents a quantitative PCR assay (5'-nuclease assay, also known as TaqMan) for quantifying small-subunit (SSU) rRNA genes (rDNAs) in mixed microbial populations. The method was applied to quantify SSU rDNAs from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations were developed for quantification at the domain and group levels, and tested for specificity and quantitative reliability. The 5'-nuclease assays reliably quantified rDNAs over at least four orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5'-nuclease assays were similar to those estimated by other methods such as quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. Ribosomal RNA genes (rDNAs) are widely used to study microbial diversity in environmental samples. Most surveys of microbial diversity in environmental samples have relied on cloning and sequencing of rDNAs. These studies have shown that the diversity of microorganisms in natural ecosystems has been severely underestimated in culture collections and have led to the discovery of several new microbial lineages. Despite increased knowledge of the phylogenetic diversity of indigenous microbes, less is known about the abundance of particular groups or their spatial and temporal dynamics. Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. PCR-based quantification methods rely on quantification after multiple replication cycles and have been shown to bias the proportions of different amplicons in mixtures. Recent studies have shown that the accumulation of amplification products may bias the proportions of different amplicons in mixtures compared to the proportions in the original samples and have suggested that a minimum number of cycles should be used. Some techniques rely on diagnostic fragment sizes, so microbial identification is presumptive, not determinative. To circumvent the quantitative difficulties associated with other methods, the study applied a recently developed PCR-based 5'-nuclease assay (also known as TaqMan) to the quantification of SSU rDNAs in artificial mixtures and in environmental DNA samples. This method detects amplified rDNAs in early cycles of the PCR and quantifies during the exponential phase of the reactions. The method also allows the estimation of PCR amplification efficiency. The study developed general 5'-nuclease assays for the measurement of rDNAs belonging to the domains Archaea and Bacteria. Specific assays were designed for the quantification of SSU rDNAs of bacteria belonging to the genera Synechococcus and Prochlorococcus and two groups of uncultivated marine Archaea. The results indicate that these assays can accurately estimate relative proportions of genes in complex mixtures. The study also revealed some of the inherent limitations of the approach. The 5'-nuclease assays were tested using artificial DNA mixturesThis study presents a quantitative PCR assay (5'-nuclease assay, also known as TaqMan) for quantifying small-subunit (SSU) rRNA genes (rDNAs) in mixed microbial populations. The method was applied to quantify SSU rDNAs from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations were developed for quantification at the domain and group levels, and tested for specificity and quantitative reliability. The 5'-nuclease assays reliably quantified rDNAs over at least four orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5'-nuclease assays were similar to those estimated by other methods such as quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. Ribosomal RNA genes (rDNAs) are widely used to study microbial diversity in environmental samples. Most surveys of microbial diversity in environmental samples have relied on cloning and sequencing of rDNAs. These studies have shown that the diversity of microorganisms in natural ecosystems has been severely underestimated in culture collections and have led to the discovery of several new microbial lineages. Despite increased knowledge of the phylogenetic diversity of indigenous microbes, less is known about the abundance of particular groups or their spatial and temporal dynamics. Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. PCR-based quantification methods rely on quantification after multiple replication cycles and have been shown to bias the proportions of different amplicons in mixtures. Recent studies have shown that the accumulation of amplification products may bias the proportions of different amplicons in mixtures compared to the proportions in the original samples and have suggested that a minimum number of cycles should be used. Some techniques rely on diagnostic fragment sizes, so microbial identification is presumptive, not determinative. To circumvent the quantitative difficulties associated with other methods, the study applied a recently developed PCR-based 5'-nuclease assay (also known as TaqMan) to the quantification of SSU rDNAs in artificial mixtures and in environmental DNA samples. This method detects amplified rDNAs in early cycles of the PCR and quantifies during the exponential phase of the reactions. The method also allows the estimation of PCR amplification efficiency. The study developed general 5'-nuclease assays for the measurement of rDNAs belonging to the domains Archaea and Bacteria. Specific assays were designed for the quantification of SSU rDNAs of bacteria belonging to the genera Synechococcus and Prochlorococcus and two groups of uncultivated marine Archaea. The results indicate that these assays can accurately estimate relative proportions of genes in complex mixtures. The study also revealed some of the inherent limitations of the approach. The 5'-nuclease assays were tested using artificial DNA mixtures