The molecular mechanism of fatty acid uptake and utilization is of high medical relevance for the treatment of obesity, diabetes, and cardiovascular disease. Neuronal processes, hormones, and transcription factors are master regulators of these essential processes while their fine-tuning is achieved by modulating the activity and amount of enzymes. Proteins involved in fatty acid uptake and metabolism are important pharmaceutical targets. Only basic research on these molecules will lead to new strategies for therapy. Conceptionally, the intracellular utilization of long chain fatty acids may be subdivided into three steps: uptake across the plasma membrane, activation by esterification with coenzyme A, and subsequent metabolism. Long chain acyl-CoA synthetases (ACSLs) activate fatty acids for intracellular metabolism but are also involved in the regulation of uptake. The predominant pathways for fatty acids are their storage, membrane biosynthesis, and conversion to energy. How activated fatty acids are channeled toward one particular metabolic pathway is not well understood on the molecular level. We have previously shown that ACSLs localized to either the endoplasmic reticulum or to mitochondria can regulate the extent of fatty acid uptake. Multiple different long chain ACSLs are expressed simultaneously in the same cell type but differ in their subcellular localization. The hypothesis we put forward here implies that the spatial organization of ACSL activity is a key factor in channeling fatty acids toward a particular metabolic fate.