GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5¢-phos-phate, the first committed intermediate in the biosynthesis of riboflavin. The enzyme was shown to contain one zinc ion per subunit. Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate fromthe imidazole ring ofGTPor from the intermediate analog, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5¢-triphosphate. .