The incorporation and distribution of 14C in 2'-deoxycoformycin, elaborated by Streptomyces antibioticus, were studied with [U-14C]glycine, [U-14C]adenosine and [U-14C]adenine. Similar ratios of 14C in the aglycon and carbohydrate portions of 2'-deoxycoformycin, ara-A, and adenosine isolated from the RNA indicated that [U-14C]adenosine was incorporated into 2'-deoxycoformycin without cleavage of the N-glycosylic bond. Following the addition of [U-14C]adenine, 98% of the 14C isolated from [14C]-2'-deoxycoformycin resided in the aglycon. 2'-Deoxycoformycin biosynthesis may not require the de novo purine biosynthetic pathway as evidenced by the failure to detect incorporation of [U-14C]glycine into 2'-deoxycoformycin. These data suggest that the biosynthesis of 2'-deoxycoformycin involves the incorporation of the carbon-nitrogen skeleton of an intact purine nucleoside or nucleotide, thereby implying that a purine ring is opened enzymatically between C-6 and N-1 and a one-carbon unit is added to form the 1,3-diazepine ring of 2'-deoxycoformycin.