Radiolabeled phosphatidate and diglyceride were prepared bound to rat liver microsomes. These compounds were used as substrates in studies of diglyceride acyltransferase, cholinephosphotransferase, and CTP:phosphatidic acid cytidylyltransferase. Optimum incubation conditions for these reactions in microsomes from normal male rats are described. High fructose diets were fed to rats for 11 days; this resulted in an increased rate of neutral lipid formation from sn-glycerol-3-phosphate by liver microsomal preparations. This was attributed, in part, to a previously reported increase in liver phosphatidate phosphatase activity. The significance of this increase is supported by the finding of a fall in microsomal phosphatidate content and a doubling in microsomal diglyceride. In addition, diglyceride acyltransferase measured with microsomal-bound diglyceride was increased twofold with no equivalent change in cholinephosphotransferase activity. Such a change should result in preferential triglyceride formation from the increased microsomal diglyceride pool. CTP:phosphatidic acid cytidylytransferase activity was depressed by the high fructose diet. These combined alterations would lead to an accelerated hepatic triglyceride formation, a result found in vivo during high fructose feeding. The high fructose diet decreased slightly the total microsomal phospholipid content and markedly depressed phosphatidylethanolamine levels.