The kinetic behavior of human serum albumin (HSA) adsorbed on a reversed-phase support was studied. With a phosphate buffer eluent (pH 7.4), the sharp elution front characterizes a fast kinetic adsorption process with a high apparent adsorption rate. In presence of 20% acetonitrile added to the eluent the apparent adsorption rate is about 60 times as low as that found for the first adsorption step in pure buffer. The largest column capacity is found with 20% acetonitrile in buffer; for larger organic solvent contents, a decrease of both the apparent adsorption rate and the column capacity are observed with increasing amounts of acetonitrile in the buffer. In order to better understand the chromatographic behavior of HSA on this type of support, we studied the structural infrared characteristics of the protein in solution. Fourier transform infrared spectra show that acetonitrile induces some structural changes of the protein in solution and competes with alkyl chains for the interaction with HSA explaining the slow adsorption kinetic process observed in presence of the organic solvent in the eluent. The more compact protein structure found with 20% acetonitrile is correlated with the larger amount of protein adsorbed at this aqueous buffer-organic solvent composition.