This study developed and tested a 5′-nuclease assay (also known as TaqMan) for quantifying specific small-subunit (SSU) ribosomal DNA (rDNA) genes from uncultivated planktonic prokaryotes in Monterey Bay. The assay was designed to quantify SSU rDNA genes at the domain and group levels, and its specificity and quantitative reliability were tested. The 5′-nuclease assay reliably quantified rDNA genes 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 assay were similar to those estimated by alternative methods such as quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. The study also discussed the limitations of the approach, particularly in designing primer and probe sets that encompass entire domains. Overall, the 5′-nuclease assay provides a valuable tool for quantifying specific prokaryotic taxa in environmental samples, with potential applications in microbial ecology and metabolic activity monitoring.This study developed and tested a 5′-nuclease assay (also known as TaqMan) for quantifying specific small-subunit (SSU) ribosomal DNA (rDNA) genes from uncultivated planktonic prokaryotes in Monterey Bay. The assay was designed to quantify SSU rDNA genes at the domain and group levels, and its specificity and quantitative reliability were tested. The 5′-nuclease assay reliably quantified rDNA genes 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 assay were similar to those estimated by alternative methods such as quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. The study also discussed the limitations of the approach, particularly in designing primer and probe sets that encompass entire domains. Overall, the 5′-nuclease assay provides a valuable tool for quantifying specific prokaryotic taxa in environmental samples, with potential applications in microbial ecology and metabolic activity monitoring.