Hepatic monoacylglycerol acyltransferase (EC 2.3.1.22) (MGAT) is a developmentally-expressed activity associated with physiological periods characterized by high rates of lipolysis and dependence on fatty acids for energy production. During these periods, MGAT may help to retain essential fatty acids selectively. In streptozotocin-diabetes, mean MGAT-specific activity increased 11.8-fold. We characterized microsomal MGAT activity from diabetic and control adult rats, and compared these adult activities with the high neonatal activity. Compared with the activity in neonatal liver, adult MGAT activity was more thermolabile, had a markedly different pH profile, and responded differently to incubation with bovine serum albumin, phospholipids, and MnCl2. Adult diabetic MGAT activity was also stimulated 2-fold by albumin and was markedly thermolabile, but was not inhibited by phospholipid. Diabetic MGAT activity had some properties that combined characteristics observed in adult and neonatal microsomes: a pH dependence that was optimal at pH 7.0 but that plateaued between pH 7.0 and 9.5, and neither stimulation nor inhibition after incubation with MnCl2. Diabetic MGAT acylated monoalkylglycerols more readily than did either the neonatal or the adult MGAT activities. The enhanced expression of hepatic MGAT activity in diabetes is consistent with its postulated role in retaining essential fatty acids during lipolysis.