Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 270 of cytosolic proteins) in the small intestine epithelium, Despite cell transfection studies, its function is still unclear, Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported, Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0.72% of cytosolic proteins in fibroblasts (Prows et al. 1997, Arch. Biochem. Biophys. 340: 135), In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell Line, Caco-2, After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of Liver FABP was selected. These cells, which had a lower I ate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocyte, Incubation of differentiated cells with [C-14]palmitate shelved a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected, A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.