The nucleotide substrate specificity of human glycinamide ribonucleotide transformylase, a chemotherapeutic target, has been examined. The enzyme accepts the sarcosyl analog of glycinamide ribonucleotide, carbocyclic glycinamide ribonucleotide, and two phosphonate derivatives of carbocyclic glycinamide ribonucleotide with V/K values, relative to that obtained for beta-glycinamide ribonucleotide, of 1, 27, 1.4, and 2.9%, respectively. Several other analogs of carbocyclic glycinamide ribonucleotide, namely a truncated phosphonate and 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-carbocyclic glycinamide ribonucleotide, were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values approximately 100 times higher than the Km for -glycinamide ribonucleotide. Although the results of the present study parallel those obtained previously with the avian enzyme (V. D. Antle, D. Liu, B. R. McKellar, C. A. Caperelli, M. Hua, and R. Vince (1996) J. Biol. Chem. 271, 6045-6049), quantitative differences between the two enzyme species have been uncovered.