The specific activities of three enzymes engaged in complex lipid synthesis, diglyceride acyltransferase, cholinephosphotransferase, and lysolecithin acyltransferase were studied in intestinal mucosa of hamsters fed either saline, hydrolyzed casein, or corn oil for 9 1/2 hr. In the most proximal intestine, saline feeding was associated with a reduced specific activity in villous tips with all three enzymes studied when compared with the two caloric supplemented groups. In the most distal intestine, oil feeding increased the activity of lysolecithin acyltransferase and choline phosphotransferase twofold as compared to casein fed hamsters; diglyceride acyltransferase was increased one- and one-half-fold. The response of lysolecithin acyltransferase and diglyceride acyltransferase to fat feeding was incomplete when compared to hamsters fed a fat supplemented diet for 7 days, suggesting that their pattern of response to dietary substrate was similar to the disaccharidases. By contrast, the response of cholinephosphotransferase to fat feeding was complete at 24 hr, suggesting that it responds in a manner similar to the glycolytic enzymes.