Malonyl-CoA sensitivity of carnitine acetyltransferase (EC 18.104.22.168) activities in isolated intact mitochondria from rat liver, heart and white and brown adipose tissue, and rabbit liver and heart were studied with a radioisotopic assay using [3H]acetyl-CoA. Malonyl-CoA inhibited [3H]acetylcarnitine formation in intact rat liver mitochondria (overt activity), while acetyltransferase in heart and white and brown adipose tissue were malonyl-CoA-insensitive. Acetyltransferase from rabbit liver and heart was only weakly inhibited by malonyl-CoA. Acetyltransferase in intact rat liver peroxisomes was malonyl-CoA-insensitive. Lysis of the mitochondria with (+)palmitoylcarnitine increased acetyltransferase activities (total activity) in all mitochondria studied and abolished malonyl-CoA-sensitivity, showing the necessity of intact mitochondrial membranes for malonyl-CoA-sensitivity. A differential increase in overt and total mitochondrial carnitine acetyltransferase was observed in rat liver upon fasting and clofibrate feeding. Fasting increased overt activity more than total activity and diminished malonyl-CoA-sensitivity. Clofibrate increased mainly total activity and also reduced malonyl-CoA-sensitivity. A similar pattern of increased activities was observed with palmitoyl-CoA and octanoyl-CoA as substrates.