Rats were fed for 35 days a high-fat diet containing either 36% of total calories as ethanol (ethanol group) or an isocaloric amount of carbohydrate (control group). Then, mitochondria were isolated from the periportal and the perivenous zone of the liver in order to study the acinar heterogeneity of the effects of prolonged ethanol administration upon the properties of carnitine palmitoyltransferase I (CPT-I) and its membrane environment. Chronic ethanol ingestion selectively decreased CPT-I activity in periportal hepatocytes but equally increased enzyme sensitivity to malonyl-CoA and enzyme energy of activation in the two zones of the liver. In control animals, mitochondrial membrane showed higher fluidity and lower degree of saturation of phospholipid fatty acyl moieties in periportal than in perivenous hepatocytes. Prolonged ethanol feeding (i) decreased mitochondrial membrane fluidity; (ii) increased the proportion of palmitic acid and decreased that of arachidonic acid in mitochondrial phosphatidylcholine and phosphatidylethanolamine, whereas it drastically reduced the content of linoleic acid and concomitantly increased that of saturated and monoenoic fatty acids in cardiolipin; (iii) suppressed the disordering effects of the addition of ethanol to mitochondrial suspensions. All these ethanol-induced alterations of membrane fluidity and fatty acyl composition were not significantly different between periportal and perivenous mitochondria. In conclusion, chronic ethanol feeding changes the activity of CPT-I in a zone-selective manner but modifies both the regulatory properties of the enzyme and the properties of its lipid environment in a non-zone-selective manner. Hence factors in addition to the properties of the mitochondrial membrane seem to be involved in the ethanol-induced alterations of the CPT-I enzyme.