Mitochondrial fatty acid oxidation disorders cause hypoglycaemia, hepatic dysfunction, myopathy, cardiomyopathy and encephalopathy. Despite their recognition for more than 15 years, diagnosis and treatment remain difficult. To help design rational diagnostic and therapeutic strategies, we studied the pathophysiology of accumulating metabolites in a whole-cell system. Acylcarnitines were quantified in cells and media of cultured fibroblasts after incubation with L-carnitine and fatty acids. Following incubation with palmitate, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD)-deficient fibroblasts compared with controls showed elevation of hydroxypalmitoyl- and palmitoyl-carnitine and reduction of C10- and shorter acylcarnitines, and following incubation with linoleate an increase in C14:2-, C18:2- and hydroxy-C18:2- acylcarnitines and reduction in C10:1-acylcarnitines. Hydroxyacylcarnitines remained more intracellular compared to corresponding saturated acylcarnitines. Incubation with decanoate and octanoate showed absence of hydroxylated acylcarnitines and correction of secondary metabolic disturbances, suggesting that optimal treatment should include medium-chain triglycerides of these chain lengths. Fibroblasts of patients with other fatty acid oxidation disorders showed distinct elevations of disease-specific acylcarnitines. This acylcarnitine analysis allows the diagnosis of LCHAD deficiency and its differentiation from other fatty acid oxidation disorders, which can pose difficulties in vivo. The strategy has allowed in-depth analysis with different substrates, providing suggestions for the rational design of treatment trials.