The binding of a series of amide derivatives of methotrexate to Lactobacillus casei dihydrofolate reductase has been studied by inhibition constant measurements and by 1H n.m.r. spectroscopy. Amide modification of the alpha-carboxylate of methotrexate was found to prevent interaction of the gamma-carboxylate with the imidazole of His 28. Estimates of the contributions to the binding energy from the alpha-carboxylate-Arg 57 and gamma-carboxylate-His 28 interactions have been made from a combination of inhibition and n.m.r. data.