The intestinal mucosa metabolizes fatty acids differently when presented to the lumenal or basolateral membrane. Expression of both liver and intestinal fatty acid binding proteins (L- and I-FABPs) uniquely in the enterocyte offers a possible explanation of this phenomenon. An organ explant system was used to analyze the relative binding of fatty acids to each protein. More fatty acid was bound to L-FABP than to I-FABPs (28% vs. 6% of cytosolic radioactivity), no matter on which side the fatty acid was added. However, a 2-3-fold increase in fatty acid binding to the intestinal paralog was noted after apical addition of palmitic or oleic acid in mucosa from chow fed rats. When oleic acid was added apically, a 1.4-fold increase in binding to I-FABP was observed in mucosa derived from chronically fat fed rats, consistent with the previously observed 50% increase in the content of that protein. Immunocytochemical localization of both FABPs in vivo demonstrated an apical cytoplasmic localization in the fasting state, and redistribution to the entire cytoplasm after fat feeding. These data are consistent with the hypothesis that I-FABP may contribute to the metabolic compartmentalization of apically presented fatty acids in the intestine.