Previous studies with cardiac myocytes from homozygous heart-type fatty acid (FA)-binding protein (H-FABP)^−/− mice have indicated that this intracellular: receptor protein for long-chain FA is involved in the cellular uptake of these substrates. Based on the knowledge that muscle FA uptake is a process highly sensitive to regulation by hormonal and mechanical stimuli, we studied whether H-FABP would play a role in this regulation. A suitable model system to answer this question is provided by H-FABP^+/− mice, because in hindlimb muscles the content of H-FABP was measured to be 34% compared to wild-type mice. In these H-FABP^+/− skeletal muscles, just as in H-FABP^−/− muscles, contents of FA transporters, i.e., 43-kDa FABPpm and 88-kDa FAT/CD36, were similar compared to wild-type muscles, excluding possible compensatory mech-anisms at the sarcolemmal level. Palmitate uptake rates were measured in giant vesicles prepared from hindlimb muscles of H-FABP^−/−, H-FABP^+/− and H-FABP^+/+ mice. For comparison, giant vesicles were isolated from liver, the tissue of which expresses a distinct type of FABP (i.e., L-FABP). Whereas in H-FABP^−/− skeletal muscle FA uptake was reduced by 42–45%, FA uptake by H-FABP^+/− skeletal muscle was not different from that in wild-type mice. In contrast, in liver from H-FABP^−/− and from H-FABP^+/− mice, FA uptake was not altered compared to wild-type animals, indicating that changes in FA uptake are restricted to H-FABP expressing tissues. It is concluded that H-FABP plays an important, yet merely permissive, role in FA uptake into muscle tissues.