Abstract Binding by the drug imipramine to the protein human serum albumin (HSA) was studied by using high-performance affinity chromatography. The association equilibrium constants and number of binding sites for imipramine with HSA were first estimated by utilizing frontal analysis. Imipramine was found to have one major binding site on HSA with an association equilibrium constant of 1.6 × 10 5 M −1 at pH 7.4 and 37 °C, as well as a second group of weaker and non-specific binding regions (8–9 mol/mol HSA) with an average association equilibrium constant of 1.5 × 10 3 M −1. Competition studies based on zonal elution were performed to identify the location of the major binding site for imipramine on HSA. Imipramine was found to have direct competition with L-tryptophan, which indicated that imipramine was interacting with Sudlow site II, or the indole-benzodiazepine site of HSA. No competition or allosteric effects were noted between imipramine and warfarin, a probe for Sudlow site I or the warfarin-azapropazone site of HSA. The association equilibrium constant found for imipramine at its site of competition with L-tryptophan also agreed with the value that was obtained for the major binding site of imipramine in the frontal analysis studies. These results confirmed that Sudlow site II was the location of the major binding site for imipramine on HSA. These results gave good agreement with previous observations made in the literature and should provide a more detailed description of how imipramine is transported in blood and of how it may interact with other drugs in the body.