The effect of malonyl-CoA on the kinetic parameters of carnitine palmitoyltransferase (outer) the outer form of carnitine palmitoyltransferase (palmitoyl-CoA: l-carnitine O-palmitoyltransferase, EC 2.3.1.21) from rat heart mitochondria was investigated using a kinetic analyzer in the absence of bovine serum albumin with non-swelling conditions and decanoyl-CoA as the cosubstrate. The K 0.5 for decanoyl-CoA is 3 μM for heart mitochondria from both fed and fasted rats. Membrane-bound carnitine palmitoyltransferase (outer) shows substrate cooperativity for both carnitine and acyl-CoA, similar to that exhibited by the enzyme purified from bovine heart mitochondria. The Hill coefficient for decanoyl-CoA varied from 1.5 to 2.0, depending on the method of assay and the preparation of mitochondria. Malonyl-CoA increased the K 0.5 for decanoyl-CoA with no apparent increase in sigmoidicity or V max. With 20 μM malonyl-CoA and a Hill coefficient of n = 2.1, the K 0.5 for decanoyl-CoA increased to 185 μM. Carnitine palmitoyltransferase (outer) from fed rats had an apparent K i for malonyl-CoA of 0.3 μM, while hat from 48-h-fasted rats was 2.5 μM. The kinetics with l-carnitine were variable: for different preparations of mitochondria, the K 0.5 ranged from 0.2 to 0.7 mM and the Hill coefficient varied from 1.2 to 1.8. When an isotope forward assay was used to determine the effect of malonyl-CoA on carnitine palmitoyltransferase (outer) activity of heart mitochondria from fed and fasted animals, the difference was much less than that obtained using a continuous rate assay. Carnitine palmitoyltransferase (outer) was less sensitive to malonyl-CoA at low compared to high carnitine concentrations, particularly with mitochondria from fasted animals. The data show that carnitine palmitoyltransferase (outer) exhibits substrate cooperativity for both acyl-CoA and l-carnitine in its native state. The data show that membrane-bound carnitine palmitoyltransferase (outer) like carnitine palmitoyltansferase purified from heart mitochondria exhibits substrate cooperativity indicative of allosteric enzymes and indicate that malonyl-CoA acts like a negative allosteric modifier by shifting the acyl-CoA saturation to the right. A slow form of membrane-bound carnitine palmitoyltransferase (outer) was not detected, and thus, like purified carnitine palmitoyltransferase, substrate-induced hysteretic behavior is not the cause of the positive substrate cooperativity.