The inhibition of total carnitine palmitoyltransferase (CPT) by short- and long-chain acylcarnitine and acyl-coenzyme A (acyl-CoA) was studied in muscle homogenates of normal controls and of five new patients with CPT deficiency using the isotope forward assay. Acetylcarnitine inhibited neither normal CPT activity nor the CPT of patients. D,L-Palmitoylcarnitine almost completely inhibited CPT in patients but only 55% of normal activity. In controls the CPT fraction sensitive to inhibition by palmitoylcarnitine appeared to be identical with the fraction sensitive to inhibition by malonyl-CoA and succinyl-CoA, which probably represents CPT II. The abnormal inhibition of CPT by palmitoylcarnitine was more likely due to product inhibition than to a detergent effect. Acetyl-CoA concentrations up to 0.4 mM and palmitoyl-CoA above optimal substrate concentrations up to 0.3 mM both inhibited normal CPT by about 25%, whereas the CPT of patients was significantly more inhibited by both substances than was normal CPT. The inhibition by acetyl-CoA was probably due to the structural relationship with malonyl-CoA and succinyl-CoA. The abnormal inhibition of CPT in patients by palmitoyl-CoA was due either to an abnormal substrate inhibition or to a detergent effect on CPT II similar to that of Triton X-100. The data indicate that in CPT deficiency total CPT activity is normal under optimal assay conditions. CPT II, however, is abnormally inhibited by fatty acid metabolites that accumulate during fasting.