In situ perfusion of the fetal side of the anesthetized rat placenta was used to monitor glucose fluxes in nondiabetic, streptozocin-induced diabetic (STZ-D), acutely hyperglycemic nondiabetic, and acutely normoglycemic STZ-D rats. STZ-D resulted in increased accumulation of glucose in the perfusate during a single passage through the fetal vasculature compared with nondiabetic rats, and this increase was maintained in normoglycemic STZ-D rats, indicating glucose release from placental stores. The fractional clearance of 3-O-[14C]methylglucose, a nonmetabolizable glucose analogue, across the placenta was decreased in both STZ-D groups compared with nondiabetic rats but unchanged in hyperglycemic nondiabetic rats, implying a reduction in glucose transporters in diabetic placentas. The difference between the transfer of D-[3H]glucose and 3-O-[14C]methylglucose indicated that 17% of the glucose was retained while traversing the placenta of nondiabetic rats, whereas a smaller percentage (8%) but a larger absolute amount (9 vs. 6 mumol/h) of glucose was retained by the placentas of the severely STZ-D rats. This retention was markedly enhanced in hyperglycemic nondiabetic rats and STZ-D rats when rendered normoglycemic. The net accumulation of perfusate glucose was less than that predicted from radiolabeled transfer data, indicating that glucose is also back transferred from the perfusate to the mother's placenta. We conclude that maternal diabetes markedly affects placental glucose flux.