The esterase-like activity of human serum albumin (HSA) toward esters of nicotinic acid was investigated under a variety of conditions such as protein concentration, temperature, pH, ionic strength, nature of buffers, and presence of organic solvents. Initial rate constants of hydrolysis of 18 nicotinates in the presence of 50 microM HSA were measured at pH 7.4 and 37 degrees C. The substrates displayed half-lifes ranging from less than 15 min (2-butoxyethyl nicotinate) to more than 95 hr (methyl nicotinate). The hydrolysis of tert-butyl nicotinate was too slow to be measurable, whereas 1-carbamoylethyl nicotinate was stabilized against hydrolysis by the presence of HSA. The rate constants of HSA-catalyzed hydrolysis were well correlated (r2 = 0.85; N = 12) with previously published data obtained in human plasma, indicating similar substrate specificities in the two biological preparations. All evidence points to serum albumin as the possible major catalyst of hydrolysis of nicotinate esters in human plasma.