CPT II-deficiency is regarded as the most common defect of lipid metabolism in skeletal muscle. A S113L mutation in CPT2 gene is found to be associated with about 90% patients. Characterization of the recombinantly produced variant His6-N-hCPT2/S113L should enable a better understanding of the pathogenesis mechanism of this disease. Human CPT II and the variant S113L were produced recombinantly. Both the wild type and the S113L variant displayed similar enzymatic activities. However, the S113L variant showed a decreased thermal stability, which was correlated with the clinical picture of the disease. In contrast to the prevailing opinion, the results of this study show that not only CPT I but CPT II is also inhibitable by malonyl-CoA. This inhibition, however, is more pronounced in patients with mutations in the CPT2 gene. Acyl-L-carnitines with more than 10 carbons were able to stabilize the mutated enzyme by pre-incubation experiments. Hence, the stabilizing effect by acyl-L-carnitines could provide a basis for possible therapeutic strategies.