Restriction maps were constructed of enzymically amplified 16S rRNA genes (rDNA) isolated from eight Clostridium species. Using maximum parsimony, a dendrogram was constructed from these and published 16S rRNA sequence data. Two distinct clusters were identified: cluster I contained C. difficile, C. sordelli, and C. bifermentans, and showed 30 of 35 restriction sites in common; cluster II contained C. tetani, C. perfringens C. sporogenes and C. botulinum C and G, and showed 20 of 35 restriction sites in common. Further analysis of cluster I organisms revealed that of five HpaII fragments, two were found in equal amounts in all organisms, one was found in varying amounts in all organisms, and two were found, in varying amounts, only in C. sordelli and C. bifermentans. C. sordelli-specific and C. bifermentans-specific HpaII fragments were demonstrated by Southern hybridization of rDNA. One HpaII site within the rDNA was present on most alleles in C. bifermentans, present on a minority of alleles in C. sordelli and absent in C. difficile. This suggested that there were two 16S rRNA alleles with different sequences present within each of the genomes of C. bifermentans and C. sordelli.