The substrate activity with both human and chicken dihydrofolate reductases (DHFR) has been examined for a series of 8-alkylpterins, 6-methyl-8-alkylpterins, and 7-methyl-8-propylpterin. All the 8-alkylpterins exhibited substrate activity with Vmax/[E]o values ranging from 1.0 to 5.4 and 2.6 to 14.8 s-1 for chicken and human DHFRs, respectively, with activity varying in the order 8-methyl > 8-allyl > 8-isopropyl > or = 8-ethyl > or = 8-propyl for both enzymes. Km values were found to range from 6.2 to 47 and 14 to 261 microM for chicken and human DHFRs, respectively, with the strength of binding varying in the order 8-propyl > 8-isopropyl > 8-ally > 8-methyl > 8-ethyl for both enzymes. Addition of a 6-methyl substituent affected the activity of the 8-alkylpterins significantly. While 6,8-dimethylpterin was a much better substrate than 8-methylpterin, the 6-methyl-8-propyl, 6-methyl-8-allyl, and 6-methyl-8-isopropyl compounds showed no substrate activity and 6-methyl-8-ethylpterin showed very weak activity with chicken enzyme only. 7-Methyl-8-propylpterin showed no substrate activity. Thermodynamic dissociation constants (Kd) for the compounds in binary complex with both human and chicken DHFRs ranged from 23 to 351 and 15 to 127 microM, respectively. Trends for the KdS were consistent with the kinetic data in suggesting stronger binding for 8-alkylpterins with larger 8-substituents. Comparison of Kd values with corresponding Km values suggested both strong cooperativity (6,8-dimethylpterin) and antagonism (6-methyl-8-isopropylpterin) with NADPH in binding to DHFR. Kd values of 20 and 10 microM for the ternary complexes of 7-methyl-8-propylpterin with human and chicken enzyme, respectively, suggest modest inhibitory activity. Application of molecular graphics modeling of ligands in the DHFR binding site has provided insight in interpreting the structure-activity relationships. The finding that different binding orientations are possible for ligands with small (8-methyl) or larger (e.g., 8-propyl) 8-substituents helps to explain the 6-methyl substituent effect and the transition from weak binding and high activity to tight binding and low activity as a function of ring-substituent pattern.