Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species, which contrasts sharply with their apparent high degree of phenotypic similarity.