The self-association in aqueous solution of folic acid (FA), 7,8-dihydrofolic acid (DHFA) and 5,6,7,8-tetrahydrofolic acid (THFA) has been studied by the use of proton magnetic resonance (1H NMR) spectroscopy. At concentrations below 10 mM, all three folates exist in (monomer)2⇌dimer equilibria with association constants (Ka) equal to 400, 66 and 14 M-1 for FA, DHFA and THFA respectively. These values decreased markedly to 157, 18 and 3 M-1, for FA, DHFA and THFA respectively, in the presence of 0.8 M KCl. The high extent of dimerization of FA is believed to impede the interaction with the active site of dihydrofolate reductase (DHFR) rendering it a poor substrate. In contrast, the DHFA with a much lower Ka is a better substrate. Conditions that lower the Ka of both FA and DHFA, (i.e., 0.8M KCl) turn them into better substrates. Based on the findings of the present study, it is also predicted that dihydro MTX may be a better inhibitor of DHFR than MTX. © 1985.