Two genes, sulB and sulC, in a folate biosynthetic gene cluster of Streptococcus pneumoniae were identified after determination of the DNA sequence between two previously reported genes, sulA and sulD, in a cloned segment of chromosomal DNA containing a mutation to sulfonamide resistance. The gene products, SulB and SulC, correspond to polypeptides of 49 and 21 kDa, respectively. SulC has GTP cyclohydrolase activity and catalyzes the first step in the folate biosynthetic pathway. SulB apparently has dihydrofolate synthetase activity in that it complements a folC mutant of Escherichia coli and thus catalyzes the last step in the pathway. Prior work showed that SulA, a dihydropteroate synthase, and SulD, a bifunctional enzyme, catalyze three intervening steps. Mapping of the mRNA transcribed from the operon was consistent with its beginning at a promoter with a -35 site (gTGtCc) and an extended -10 site (T-TG-TAaAAT) and its termination at the end of a hairpin structure, which would give a transcript 3,745 nucleotides in length. SulC showed a considerable conservation of sequence by comparison with proven or putative GTP cyclohydrolases from four unrelated species, with 38 to 53% of the residues being identical. A similar comparison of SulB with dihydrofolate synthetases showed an identity of only 26 to 37%. Overall, comparisons of the five folate biosynthetic enzymes in different species suggest that S. pneumoniae is related more closely to other gram-positive bacteria, less closely to eucaryotes, and least closely to the gram-negative E. coli. The varied arrangements of folate biosynthetic genes in different species imply an early evolutionary period of fluidity in genomic rearrangement.