We have investigated the effects of fatty acids on the Na+-H+ exchanger and other carrier-mediated transport systems in intestinal brush-border membrane vesicles. The Na+-H+ exchanger (i.e. H+ gradient-dependent, dimethylamiloride-sensitive Na+ uptake) was strongly inhibited by fatty acids and the inhibition was concentration dependent. Unsaturated fatty acids showed more inhibition than saturated fatty acids. Among unsaturated fatty acids, ricinoleic acid was found to be the most potent inhibitor. Inhibition of the Na+-H+ exchanger by oleic acid was partially reversible, and the nature of the inhibition was found to be non-competitive with respect to Na+. The dimethylamiloride-sensitive Na+ uptake measured in the absence of an H+ gradient was also inhibited by oleic acid, suggesting that the inhibition of the Na+-H+ exchanger by fatty acids was not due to the accelerated dissipation of the H+ gradient. Treatment of the membrane vesicles with oleic acid also inhibited other carrier-mediated transport systems as well, such as the H+ gradient-driven transport of glycylsarcosine and the Na+ gradient-driven transport of D-glucose and L-alanine, whereas it did not affect the permeability of L-glucose, a non-carrier-mediated process. However, the inhibitory effects of oleic acid on the transport of D-glucose and L-alanine appeared to be related to the enhanced collapse of the Na+ gradient rather than a direct effect on the carrier systems because transport of these solutes when measured in the absence of a Na+ gradient ([Na+]i = [Na+]o) was not affected by oleic acid. These data demonstrate that fatty acids bring about significant alterations in the activities of various transport systems of the small intestinal brush-border membrane, either by directly interacting with the transport protein or by abolishing the energy source that is necessary for the transport process.