Multiple binding of laurate (n-dodecanoate) to human serum albumin was studied by a kinetic dialysis method. With this method the concentration of unbound ligand can be determined by measuring the rate of exchange of radioactive label across a dialysis membrane under conditions of equilibrium. Determination of the rate constant of exchange, in the absence of albumin, revealed that laurate does not change its state of aggregation over a concentration range from 0.1 microM to 500 microM, indicating the prevalence of a monomer. Binding of laurate to albumin, at pH 7.5, 37 degrees C, was investigated through 220 determinations of binding equilibria in the range of 0-10 mol laurate/mol albumin, corresponding to a concentration range of unbound laurate from 1 nM to 0.1 mM. Binding data were analyzed in terms of stepwise binding. Considering the stochastic errors of the experimental data, we generated a variety of possible (equal goodness of fit) solutions to the stoichiometric binding equation. This analysis resulted in reasonably well-defined values for the first two step constants, with an indication of negative interaction. The variation of the higher step constants excluded any mechanistic conclusions, although the binding isotherms, defined by these varying sets of binding constants, fitted the experimental data well within a chosen probability limit.