Despite decades of extensive research, the transport routes, mechanisms of uptake and points of flux control of long-chain fatty acids (FA) in mammalian organs are still incompletely understood. In non-fenestratred organs such as heart and skeletal muscle, membrane barriers for blood-borne FA are the luminal and abluminal membranes of endothelial cells, the sarcolemma and the mitochondrial membranes. Transport of FA through the phospholipid bilayer of the cellular membrane is most likely accomplished by diffusion of protonated FA. Evidence is accumulating that membrane-associated proteins, such as plasmalemmal fatty acid-binding protein (FABPpm) and fatty acid translocase (FAT/CD36), either alone or in conjunction with albumin binding protein (ABP), are instrumental in enhancing the delivery of FA to the cellular membrane. Inside the cell, cytoplasmic fatty acid-binding proteins (FABPc) are involved in diffusion of FA from the plasmalemma to the intracellular sites of conversion, such as the mitochondrial outer membrane. After conversion of FA to FACoA, the fatty acyl chain is transported across the mitochondrial inner membrane in a carnitine-mediated fashion. Uptake and utilization of FA by muscle cells are finely tuned, most likely to avoid the intracellular accumulation of FA, as these are cytotoxic at high concentrations. On a short-term basis, net uptake is, among others, regulated by intracellular translocation of FAT from intracellular stores to the sarcolemma and by the concentration gradient of FA across the sarcolemma. The latter implies that, among others, the rate of FA utilization determines the rate of uptake. The rate of utilization is governed by a variety of factors, including malonylCoA, the ratio acetylCoA/CoA and the availability of competing substrates such as glucose, lactate, and ketone bodies. Long-term regulation of uptake and utilization is accomplished by alterations in the rate of expression of genes, encoding for FA-handling proteins. Circumstantial evidence indicates that FA themselves are able to modulate the expression of FA-handling genes via nuclear transcription factors such as peroxisome proliferator-activated receptors (PPARs).