Fatty acid translocase (FAT/CD36) is a membrane protein putatively involved in the transmembrane transport of long-chain fatty acids. We tested the hypothesis that expression of this protein in H9c2, a rat heart cell line normally not expressing FAT, would increase cellular palmitate uptake. We were able to stably transfect H9c2 cells with FAT, yielding 15 cell lines showing varying levels of FAT expression. The uptake and metabolism of palmitate was first studied in the non-transfected H9c2 cells and in two FAT-transfected cell lines. In each case, uptake of palmitate was found to be linear in time for at least 30 min and the uptake rate was saturable with increasing palmitate concentrations. Using conditions under which the maximal capacity of intracellular palmitate handling was not fully utilized, we tested 7 out of 15 FAT-transfected cell lines with varying FAT expression levels. No significant correlation was found between the level of FAT expression and the rate of palmitate uptake. In conclusion, we found that palmitate uptake by H9c2 cells occurs mainly by passive diffusion. Fatty acid translocase (FAT) transfection did not significantly increase the palmitate uptake rate, raising the possibility that H9c2 cells lack a protein (or set of proteins) that acts as an obligatory partner of FAT in long-chain fatty acid transport from the extracellular compartment to the cytoplasm.