GTP cyclohydrolase II (GCH II) catalyzes the first step in the biosynthesis of riboflavin. GCH II catalyzes the opening of the purine ring of GTP to produce 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. Streptomyces coelicolor maintains three GCH II homologs, each of which is encoded in a distinct gene context. UV-VIS spectroscopy, mass spectrometry, and HPLC have been used to show that two of the enzymes catalyze the canonical reaction while the third produces 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. Site directed mutagenesis studies show that a single amino acid change in the active site is sufficient to interconvert the catalytic activities of all three homologs, suggesting that this residue is a switch residue. A mechanistic basis for the switch is proposed. This research is supported (in part) by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (V. B.). Additional support for V. B. from NIH (GM 72623) is gratefully acknowledged. J. E. S. is supported by a NIH predoctoral training grant (GM 08804).