Fatty acid uptake and metabolism are often dysregulated in cancer cells. Fatty acid activation is a critical step that allows these biomolecules to enter cellular metabolic pathways such as mitochondrial β-oxidation for ATP generation or the lipogenic routes that generate bioactive lipids such as the inositol phospholipids. Fatty acid activation by the addition of coenzyme A is catalysed by a family of enzymes called the acyl CoA synthetase ligases (ACSL). Furthermore, enhanced expression of particular ACSL isoforms, such as ACSL4, is a feature of some more aggressive cancers and may contribute to the oncogenic phenotype. This study focuses on ACSL3 and ACSL4, closely related structural homologues that preferentially activate palmitate and arachidonate fatty acids, respectively. In this study, immunohistochemical screening of multiple soft tissue tumour arrays revealed that ACSL3 and ACSL4 were highly, but differentially, expressed in a subset of leiomyosarcomas, fibrosarcomas and rhabdomyosarcomas, with consistent cytoplasmic and granular stainings of tumour cells. The intracellular localisations of endogenously expressed ACSL3 and ACSL4 were further investigated by detailed subcellular fractionation analyses of HT1080 fibrosarcoma and MCF-7 breast cancer cells. ACSL3 distribution closely overlapped with proteins involved in trafficking from the trans-Golgi network and endosomes. In contrast, the ACSL4 localisation pattern more closely followed that of calnexin which is an endoplasmic reticulum resident chaperone. Confocal immunofluorescence imaging of MCF-7 cells confirmed the intracellular localisations of both enzymes. These observations reveal new information regarding the compartmentation of fatty acid metabolism in cancer cells.