A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolating the 16S rRNA or cloning its gene. The method uses reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences to selectively target the 16S rRNA in bulk cellular RNA. Three particularly useful priming sites provide access to the three major 16S rRNA structural domains and routinely yield 800–1000 nucleotides of 16S rRNA sequence. The method is evaluated for accuracy, sensitivity to modified nucleotides, and phylogenetic usefulness. The approach is simple and should facilitate the rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses. The classification of organisms has traditionally been based on morphological, developmental, and nutritional characteristics. However, with microorganisms, these criteria do not necessarily correlate well with natural evolutionary relationships. Molecular phylogenetic analysis is now widely used for describing microbes, both eukaryotic and prokaryotic. Available molecular methods for evaluating phylogenetic relationships have advantages and limitations. Macromolecular sequences are preferred because they allow quantitative inference of relationships. Ribosomal RNAs, particularly 16S rRNA, have proven the most useful for establishing distant relationships due to their high information content, conservative nature, and universal distribution. The method described here rapidly provides partial sequences of 16S rRNA that are useful for phylogenetic analysis. The approach uses reverse transcriptase to generate sequences from RNA templates. The method involves purifying RNA templates, synthesizing oligodeoxynucleotide primers, and performing reverse transcription reactions. The results show that the method is accurate and effective for generating sequence data. The method is applicable to a wide range of organisms, including eubacteria, archaebacteria, and eukaryotes. The primers used are operationally termed "universal" because they work effectively for a wide range of organisms. The method is simple and efficient, allowing for the rapid generation of sequence data. The approach is amenable to the screening of large numbers of organisms, making it a valuable tool for phylogenetic analysis. The method provides rapid and convenient access to the appropriate sequence information for phylogenetic studies. The results show that the method is effective for generating sequence data and is applicable to a wide range of organisms. The method is a valuable tool for phylogenetic analysis and could become a standard tool for laboratories concerned with characterizing organisms of uncertain affiliation.A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolating the 16S rRNA or cloning its gene. The method uses reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences to selectively target the 16S rRNA in bulk cellular RNA. Three particularly useful priming sites provide access to the three major 16S rRNA structural domains and routinely yield 800–1000 nucleotides of 16S rRNA sequence. The method is evaluated for accuracy, sensitivity to modified nucleotides, and phylogenetic usefulness. The approach is simple and should facilitate the rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses. The classification of organisms has traditionally been based on morphological, developmental, and nutritional characteristics. However, with microorganisms, these criteria do not necessarily correlate well with natural evolutionary relationships. Molecular phylogenetic analysis is now widely used for describing microbes, both eukaryotic and prokaryotic. Available molecular methods for evaluating phylogenetic relationships have advantages and limitations. Macromolecular sequences are preferred because they allow quantitative inference of relationships. Ribosomal RNAs, particularly 16S rRNA, have proven the most useful for establishing distant relationships due to their high information content, conservative nature, and universal distribution. The method described here rapidly provides partial sequences of 16S rRNA that are useful for phylogenetic analysis. The approach uses reverse transcriptase to generate sequences from RNA templates. The method involves purifying RNA templates, synthesizing oligodeoxynucleotide primers, and performing reverse transcription reactions. The results show that the method is accurate and effective for generating sequence data. The method is applicable to a wide range of organisms, including eubacteria, archaebacteria, and eukaryotes. The primers used are operationally termed "universal" because they work effectively for a wide range of organisms. The method is simple and efficient, allowing for the rapid generation of sequence data. The approach is amenable to the screening of large numbers of organisms, making it a valuable tool for phylogenetic analysis. The method provides rapid and convenient access to the appropriate sequence information for phylogenetic studies. The results show that the method is effective for generating sequence data and is applicable to a wide range of organisms. The method is a valuable tool for phylogenetic analysis and could become a standard tool for laboratories concerned with characterizing organisms of uncertain affiliation.