A carnitine palmitoyltransferase (CPT), extracted from microsomes with octyl glucoside, was purified and characterized as a 54-kDa protein and was found to show no malonyl-CoA inhibition (Murthy, M. S. R., and Bieber, L. L. (1992) Protein Exp. Purif. 3, 75-79). We show here that the malonyl-CoA-sensitive CPT of microsomes associates with their membrane, whereas the above 54-kDa CPT is a soluble luminal protein. Western blot probing with antibody to the 54-kDa CPT was found to show a positive response with the soluble microsomal fraction but not with their membranes. 2-Tetradecylglycidyl-CoA inhibited the membrane-associated CPT activity irreversibly, whereas the inhibition of the soluble CPT was largely reversible. Exposure of microsomes to [3H]etomoxir, ATP, and CoA led to the labeling of a approximately 47-kDa peptide that associated with membranes, whereas no such peptide labeling was seen with the soluble microsomal fraction. These and other results show (a) that microsomes have malonyl-CoA-sensitive, as well as malonyl-CoA-insensitive, CPT activities, (b) that these two activities are due to distinct proteins, (c) that the malonyl-CoA-sensitive CPT of microsomes is a previously uncharacterized CPT isoform, and (d) that the [3H]etomoxir-labeled approximately 47-kDa peptide is a likely candidate for the microsomal malonyl-CoA-sensitive CPT or its regulatory subunit.