A post 30,000 x g particulate fraction was isolated from rat heart. This mixed membrane fraction is enriched in a carnitine palmitoyltransferase which is sensitive to both malonyl-CoA and etomoxiryl-CoA at concentrations that inhibit the malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo/CPT-I) of intact mitochondria. Tritiated etomoxiryl-CoA labels two proteins with the same molecular weight as the labeled proteins from rat heart mitochondria. Malonyl-CoA-sensitive carnitine palmitoyltransferase in the particulate fraction is stable to freeze-thawing, and the activity is not latent. These data show that the carnitine palmitoyltransferase associated with this particle is CPTo/CPT-I. Positive Western blots were obtained, with the particle using anti-CPTi/CPT-II at a molecular weight identical with the CPT1/CPT-II purified from rat heart mitochondria. Catalytic activity was purified to near homogeneity in approximately 40% yield. The purified protein has a molecular weight identical with CPTi/CPT-II, it cross-reacts with antibody against CPTi/CPT-II, it is not inhibited by malonyl-CoA or etomoxiryl-CoA, and mass spectral analyses of the tryptic peptides give the same molecular masses as CPTi/CPT-II, and, when mixed with equal amounts of CPTi/CPT-II, one uniform spot is found by two-dimensional electrophoresis. These data indicate that the catalytic subunit of CPTo/CPT-I is the same as CPTi/CPT-II. The average inhibition of the CPT of frozen-thawed particles is 71% by 50 nM etomoxiryl-CoA and 62% by 50 nM malonyl-CoA. The inhibitor sensitivity, but not the catalytic activity, is lost by solubilization in 1% Triton X-114; removal of Triton X-114 using Extracti-Gel D restores etomoxiryl-CoA and malonyl-CoA sensitivity (both 50 nM) of CPT to an average of 77 and 48%, respectively. Consistent with previous reports, these results show that CPTo/CPT-I is NOT inactivated by detergents, rather detergents both desensitize it to malonyl-CoA and alter its Vmax. These data show the assumption that CPTo/CPT-I is inactivated by detergents is untenable.