Acylcarnitines are important diagnostic markers for inborn errors of fatty acid oxidation, but their analysis in body fluids may not always be reliable. Recently, disease-specific acylcarnitine profiles generated by cultured skin fibroblasts were reported to facilitate the diagnosis by localizing a specific enzymatic defect in the mitochondrial beta-oxidation pathway. Using a novel methodologic approach, fibroblasts from 16 patients with inborn errors of fatty acid oxidation and 13 control subjects were preincubated with L-[3H]carnitine to label the intracellular carnitine pool. Cells were subsequently incubated with unlabeled palmitic acid and, after methanol extraction of cells and media, labeled free carnitine and acylcarnitines were analyzed by radio-HPLC. Quantitation was based on the integrated radioactivity of individual peaks relative to the total radioactivity recovered. In control cell lines, all saturated acylcarnitines were detected, and reference values were established. With the exception of one cell line deficient in electron transfer flavoprotein, all mutant cell lines showed abnormal and disease-specific relative concentrations of acylcarnitines. Advantages of the method include use of a small number of cells, no need for trypsinization and permeabilization of cells before incubation, simple extraction without purification of the specimen before HPLC, and relatively inexpensive equipment. The method allows a focused approach to the subsequent, more laborious confirmation of a particular disease by direct enzymatic and/or molecular analysis. It remains to be established whether the method can replace widely used global measurements of fatty acid oxidation rates in vitro that do not provide specific information about the enzyme deficiency involved.