Affinities of long chain fatty acids (FA) for fatty acid-binding proteins (FABPs) have been measured by monitoring the concentrations of the unbound or free fatty acids (FFA) in equilibrium with the FABPs using the fluorescent probe ADIFAB. This probe allows the measurement of the concentration of FFA in equilibrium with FABPs, without physical separation of any of the reactants. Equilibrium characteristics were measured at 37 degrees C for palmitate, stearate, oleate, linoleate, linolenate, and arachidonate binding to six FABPs from intestine, heart, adipose, and liver from different species. Equilibrium constants for each FA were found to be extremely sensitive to the tissue origin of the FABP but largely independent of species differences. The measured values of the dissociation constants (Kd) ranged from about 2 to 1000 nM, depending upon the tissue origin of the FABP and the FA. Binding constants for some FABPs varied considerably with different FA, as much as 80-fold in the case of the intestinal FABP. In contrast, Kd values for adipocyte FABPs exhibited less than 4-fold variation with FA type and are generally larger (lower affinities) than for the other FABPs. For all FABPs, Kd values for fatty acids with the same chain length were considerably lower for saturated as compared to polyunsaturated FA. This characteristic likely reflects the lower aqueous solubilities of the saturated fatty acids. In contrast to the other FABPs, rat liver FABP was found to have two FA-binding sites/monomer. Each of these two sites had similar high affinities for the saturated FA, while for the unsaturated FA the two sites exhibited affinities that differ by more than 7-fold. This study disagrees with earlier investigations in finding that equilibrium binding of FA to FABPs is a sensitive function of FA type and FABP tissue origin and that FA-FABP dissociation constants are submicromolar. These results provide a framework with which to understand better the biological function of FABPs and the FA-FABP interaction.