Intestinal mucosal microsomes from several animal species (non-fasted) were used to study the formation of glycerolipids from sn-[1,3-14C]glycerol 3-phosphate and palmitoyl-CoA. Rates of glycerolipid biosynthesis were species dependent, since mouse and rat were quite low compared to hamster, guinea pig and man. Under the usual incubation conditions, guinea pig intestinal microsomes formed primarily phosphatidic acid (85-90%). However, when Mg2+ was added to incubations containing intestinal microsomes prepared in the presence of 5 mM EDTA, there was a marked increase in neutral lipid biosynthesis (5-10-fold). These results suggest that intestinal microsomes contain mg2+-dependent phosphatidate phosphohydrolase (EC 3.1.3.4) which is involved in glycerolipid biosynthesis. All divalent cations studied increased sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity; however, Ca2+, Ba2+, Zn2+, Cd2+, Ni2+, and Mn2+ antagonized the Mg2+-dependent rise in neutral lipid formation. In all cellular preparations studied, the ratio of neutral lipid (diacylglycerol + triacylglycerol) to total lipid (phosphatic acid + neutral lipid) was low, suggesting that phosphatidate phosphohydrolase may be rate limiting in intestinal neutral glycerolipid biosynthesis.