Thermodynamic dissociation constants (Kd) have been determined for two series of 8-alkyl-N5-deazapterins in binary complexes with human and chicken dihydrofolate reductases (DHFRs) and ternary complexes with the enzyme.NADPH complex. For an initial series of 12 compounds with variable 8-alkyl substitutents and pyrazine ring-methyl substitution patterns, Kd values at pH 6.6 were found to range from > 100 to 0.5 microM, with consistent trends depending on the enzyme source, the size of the 8-substituent, and the presence and position of the pyrazine ring-methyl substituent. For most compounds in this first series, Kd values were significantly lower for the ternary complex than for the binary complex with ratios of Kd(binary)/Kd(ternary) ranging from 0.6 to 62, suggesting a degree of cooperativity in binding to the enzyme between ligand and cofactor. This effect was more pronounced for the human enzyme. The structure-activity relationships developed in the first series suggested a number of strategies for developing ligands with greater affinity for DHFR. These were tested with a second series of four compounds. The Kd of 80 nM at pH 6.6 of one of these compounds [5-methyl- 8-isobutyl-N5-deazapterin (15)] in ternary complex with human DHFR is more than 200 times lower than that for the lead compound (8-methyl-N5-deazapterin (1); Kd 21 microM). Studies of binding stoichiometry indicated two binding sites in binary complexes with DHFR for 8-alkyl-N5-deazapterins with smaller 8-substituents. The second site was not found in ternary complexes or for ligands with larger 8-substituents, suggesting that the second ligand molecule in binary complexes is probably binding in the cofactor site and that the larger 8-substituents also bind in this area. A detailed study of the inhibition kinetics for one compound, 6,8-dimethyl-N5-deazapterin (5), showed it to be a competitive inhibitor of the chicken DHFR-catalyzed reduction of 6,8-dimethylpterin suggesting that the 8-alkyl-N5-deazapterins bind in the substrate site of DHFR. The pH dependence of the binding of several ligands in binary and ternary complexes with DHFR was examined by determining their Kd values at a range of pH's. This suggested that binding was predominantly between protonated ligand and deprotonated enzyme, but with variable contributions to binding observed between deprotonated enzyme and neutral ligand, and protonated enzyme and protonated ligand, depending on compound and complex type.