Formycin A is a potent purine nucleoside antibiotic with a C-glycosidic linkage between the ribosyl moiety and the pyrazolopyrimidine base. Herein, a cosmid is identified from the Streptomyces kaniharaensis genome library that contains the for gene cluster responsible for the biosynthesis of formycin. Subsequent gene deletion experiments and in vitro characterization of the forBCH gene products established their catalytic functions in formycin biosynthesis. Results also demonstrated that PurH from de novo purine biosynthesis plays a key role in pyrazolopyrimidine formation during biosynthesis of formycin A. The participation of PurH in both pathways represents a good example of how primary and secondary metabolism are interlinked.