The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stern (ES) cells stably expressing intestinal fatty acid binding protein (IFABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7nitrobenz-2-oxa-1,3- diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (D(eff)) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and D(eff) by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I- FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.