It has recently been hypothesized that fatty acid (FA) transfer across the myocardial capillary wall is mediated by cytoplasmic fatty acid-binding protein (FABP). Therefore, we studied the type and content of FABP in endothelial cells from rat heart, using molecular biological, immunochemical, and FA-binding assays. Studies were performed on short term cultured endothelial cells, two established endothelial cell lines and ultrathin cryosections from adult rat heart. Northern blotting analysis of endothelial cell RNA failed to detect either heart-type (H-) FABP or liver-type (L-) FABP mRNA, but the reversed transcription-polymerase chain reaction revealed both H- and L-FABP mRNAs, indicating the presence of minor amounts of these mRNAs. Highly sensitive immunochemical assays (sandwich ELISAs) using specific antibodies raised against rat H- or L-FABP showed the contents of these FABP-types in endothelial cells to be 1-5 ng/mg cytosolic protein, which is more than three orders of magnitude lower than the contents of H-FABP in heart or L-FABP in liver. Immuno-electron microscopy also showed that the concentration of H-FABP in endothelial cells is at least two orders of magnitude lower than that in cardiomyocytes. Finally, cytosolic protein samples from endothelial cells revealed no significant FA-binding activity in the 15-kDa region. We conclude that rat heart endothelial cells contain only minor quantities of cytoplasmic FABP and that, therefore, FA transport over the endothelium is mediated by FABP only to a minor extent. It is postulated that aqueous diffusion of FA through the endothelial cytoplasm most likely accounts for the experimentally observed rates of cardiac FA utilization.