The maximal activity of the overt from of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) and its sensitivity to inhibition by malonyl CoA were measured in mitochondria prepared from the livers of rats which had been fed for 10 weeks on either a low fat diet (LF; 2.4% fat by weight) or on one of four high fat diets which contained 20% by weight of either hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO) or menhaden (fish) oil (MO). CPT I activity (i.e. activity per g of liver tissue), was elevated in animals fed the OO, SO or MO diets compared with those fed the LF or HCO diets. Feeding the HCO diet did not result in elevation of CPT I activity compared with feeding the LF diet. CPT I specific activity (i.e. activity per mg mitochondrial protein) was elevated in animals fed SO diet, but not in animals fed any of the other high fat diets. These observations suggest that an elevated fat load is not solely responsible for increasing CPT I activity, but that the fatty acid composition of the diet also plays a role. Hepatic CPT I activity of rats fed the LF diet was most sensitive to inhibition by malonyl CoA ([I50] = 0.53 microM). Each of the high fat diets decreased the sensitivity of CPT I to inhibition by malonyl CoA; CPT I activity in the livers from animals fed the MO diet was the least sensitive to malonyl CoA inhibition ([I50] = 1.8 microM). The fatty acid compositions of the major mitochondrial membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine and cardiolipin were modified according to the fatty acid composition of the diet. Each of these phospholipids had a distinct fatty acid composition and similar effects of dietary lipid manipulation on the fatty acid compositions were observed. Feeding the SO diet resulted in fatty acid compositions which were most similar to those found after feeding the LF diet. Feeding the HCO and OO diets increased the proportions of stearic and oleic acids, respectively, while decreasing the proportion of linoeic acid. Feeding the MO diet resulted in increased proportions of palmitic, palmitoleic, eicosapentaenoic and docosahexaenoic acids and decreased proportions of linoleic and arachidonic acids in each of the phospholipids. It is proposed that the effects of dietary lipid manipulation upon CTP I activity and sensitivity to inhibition by malonyl CoA are due to alterations in the fatty acid composition of the phospholipids in the mitochondrial membrane where CPT I resides.