Carnitine palmitoyltransferases (CPT), located both in the outer (CPT I) and inner membrane (CPT II) of mitochondria, are the key players for an efficient transport of long chain fatty acids into this cell compartment. The metabolite malonyl-CoA is known to inhibit CPT I, but not CPT II. His6-N-hCPT2 (wild type) and His6-N-hCPT2/ S113L (variant) were produced recombinantly in prokaryotic host, purified and characterized according to their functional and regulatory properties. The wild type and the variant showed the same enzymatic activity and were both inhibited by malonyl-CoA and malonate in a time-dependent manner. The inhibition was, however, significantly more pronounced in the mutated enzyme. The residual activities were 40% and 5% at temperatures of 4 °C and 30 °C, respectively. The inhibitory effect proceeded irreversibly with no recovery after postincubation of palmitoyl-CoA (Pal-CoA) as native substrate. A model of malonyl-CoA and malonate binding to human CPT II was suggested by docking studies to explain the action of the inhibitors regarding to the effect of the mutation on the protein conformation. Results indicated that not only CPT I, but also CPT II can be inhibited by malonyl-CoA. Thus, the complete inhibition of total CPT (i.e. CPT I and CPT II) in muscle homogenates by an established assay is not due to a lack of enzymatically active CPT II, but rather due to an abnormal regulation of the enzyme.