The large copy number of rRNA makes it an appealing target for oligonucleotide probes designed to identify microorganisms. Given that nucleotide sequences in rRNA are known to reflect phylogeny, species-specific rRNA probes should be feasible if the sequences found in closely related species are different. We sequenced portions of the 16S rRNA of three closely related clostridia found in the human colonic microflora: Clostridium bifermentans, C. sordellii, and C. difficile. The rRNAs of these three species showed 97 to 98% sequence similarity. Five oligonucleotide probes complementary to unique segments of the sequences were end labeled with 32P and hybridized on a nylon filter to the immobilized rRNA of each clostridium. Each probe efficiently hybridized only to the rRNA of the species to which it was directed. Complementary probes emitted a signal that exceeded by a factor of 100 to 1,000 the signal of probes that mismatched the target rRNA by 2 to 5 bases. Even a 1-base difference in rRNA sequence allowed a clear distinction between species. A systematic approach can efficiently yield taxon-specific oligonucleotide probes directed at rRNA.