The previous identification of a membrane fatty acid binding protein (MFABP) in brush border plasma membranes of the jejunum suggested that mucosal cell uptake of fatty acids might represent a carrier-mediated transport system. For evaluation of this hypothesis cellular influx kinetics (V0) of [3H]-oleate were examined in isolated rat jejunal mucosal cells. With increasing unbound oleate concentration in the medium V0 was saturable (Km = 93 nM; Vmax = 2.1 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Pretreatment of the cells with a monospecific antibody to MFABP significantly inhibited V0 of oleate, other long-chain fatty acids, and D-monopalmitin, but not of L-alanine. Moreover, in the in vivo system of isolated perfused jejunal segments the physiologic significance of MFABP in the directed overall intestinal absorption process of fatty acids was documented. In the presence of the anti-MFABP oleate absorption was markedly reduced, whereas uptake of L-alanine remained unaltered. By antibody inhibition studies it was suggested that this membrane carrier also reveals transport competence for various other long-chain fatty acids, D-monopalmitin, L-lysophosphatidylcholine, and cholesterol. These data support the hypothesis that absorption of fatty acids is mediated by a fatty acid binding membrane protein.