Several analogs of glycinamide ribonucleotide and phosphoribosylamine have been prepared and evaluated as substrates for glycinamide ribonucleotide synthetase purified from chicken liver. Glycinamide ribonucleotide analogs include side chain modifications wherein the glycine side chain (R = CH2NH2) has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, ribose ring replacement by chiral cyclopentane and cyclopentene derivatives, and phosphate replacement by phosphonates. All of these, with the exception of the O-phosphonate, served as substrates for the reverse enzymatic reaction, with V(max) values comparable to that obtained with glycinamide ribonucleotide, although the K(m) values ranged from 21 to 118 times the K(m) for glycinamide ribonucleotide. Analogs of phosphoribosylamine examined as substrates for the forward reaction consist of chiral derivatives of cyclopentane and cyclopentene and a chiral carbocyclic phosphonate. These also served as substrates, with K(m) values ranging from 5 to 23 times the K(m) for phosphoribosylamine and with diminished V(max) values. These studies have begun to define the structural features of the nucleotide substrate necessary to support enzymatic activity. Sarcosine (N-methylglycine) and β- alanine were also accepted as substrates, albeit with reduced affinity compared with glycine.