The activity and the kinetic properties of hydroxymethylglutaryl-CoA reductase and of acyl-CoA:cholesterol acyltransferase in the liver microsomal fraction have been compared between rats fed on either unsaturated or on saturated fat. When rats wre fed for 12h on a compounded diet containing either safflower seed oil or tristearin the composition of the fatty acyl chains of the microsomal phospholipids was shown to be relatively more unsaturated in the rats that received the unsaturated fat. The activity of hydroxymethylglutaryl-CoA reductase in the microsomal fraction was considerably reduced in rats fed on compounded diet containing unsaturated fat whereas this dietary condition resulted in a considerable increase in the activity of acyl-CoA:cholesterol acyltransferase. Similar effects were observed after feeding rats for 12 h on a commercial diet supplemented with either safflower seed oil or with tristearin. The addition of 2% cholesterol to the fat-supplemented diets resulted in both cases in a decrease in hydroxymethylglutaryl-CoA reductase and an increse in acyl-CoA:cholesterol acyltransferase activity as compared with the corresponding values from the rats fed on the fat-supplemented diets with no cholesterol. The Arrhenius plots of hydroxymethylgutaryl-CoA reductase in the microsomal fraction from rats fed on fat-supplemented commercial diet for 12 h showed breaks in the activation energy at 29.6 degrees C for the preparations from rats fed on tristearin and 28 degrees C for those from rats fed on safflower seed oil. The activation energy of the enzyme was lower above and higher below the break for the preparations from rat fed on the unsaturated fat-supplemented diet. Similar differences were obtained from the comparison of the Arrhenius plots in the preparations from rats fed on saturated fat and those in the preparations from rats fed on unsaturated fat when the diet was compounded and given to the animals for 36 h. The addition of 2% cholesterol to the commercial diet supplemented with either saturated or unsaturated fat resulted in Arrhenius plots with a constant activation energy between 37 and 22 degrees C for the enzyme in microsomal preparations from both groups of rats. The apparent Km value for hydroxymethylglutaryl-CoA was lower for the reductase in microsomal preparations from rats fed on the unsaturated fat as compared with that for the enzyme in microsomal preparations from rats fed on saturated fat. There was also a decrease in the apparent Km value for oleic acid for the acyltransferase from rats fed on unsaturated fat as compared with that for the enzyme in the microsomal preparation from the rats fed on saturated fat. The results of the present study are consistent with higher concentration of free cholesterol in endoplasmic reticular membrane in the environment of the reductase and that of acyltransferase following the administration of dietary unsaturated fat as compared with that following the administation of saturated fat.