Previously we reported that transposon Tn917 mutagenesis of Streptococcus mutans JH1005 yielded an isolate detective in its normal ability to produce a mutacin (P. J. Crowley, J. D. Hillman, and A. S. Bleiweis, abstr. D55, p. 258 in Abstracts of the 95th General Meeting of the American Society for Microbiology 1995, 1995). In this report we describe the recovery of the mutated gene by shotgun cloning. Sequence analysis of insert DNA adjacent to Tn917 revealed homology to the gene encoding formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine. Analysis of the fhs mutant grown anaerobically in a minimal medium demonstrated that the mutant had an absolute dependency only for adenine, although addition of methionine was necessary for normal growth. Coincidently it was discovered that the mutant was sensitive to acidic pH; it grew more slowly than the parent strain on complex medium at pH 5. Complementation of the mutant with an integration vector harboring a copy of fhs restored its ability to grow in minimal medium and at acidic pH as well as to produce mutacin. This represents the first characterization of Fhs in Streptococcus.