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 mechanisms 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.