ABSTRACT.The concentration differences across the pregnant uterus of glucose, lactate, ketoacids, free fatty acids (FFA), and oxygen were determined in 21 chronically catheterized guinea pigs. Polyvinyl catheters were inserted into one of the ovarian veins and the right carotid artery around the 50th day of pregnancy. Postoperative recovery of maternal substrate concentrations in this preparation was evaluated in five animals. Elevated hemoglobin and ketoacid concentrations persisted up to the 4th day after surgery in the awake animal. In 16 animals between 54 and 62 days gestation, arterial and venous blood samples were collected between the 4th and 13th postoperative days. Arteriovenous substrate differences across the pregnant uterus (means ± SD) were as follows: glucose, 0.87 ± 0.22 mM; lactate, 0.31 ± 0.11 mM; and oxygen 4.77 ± 0.58 mM. There was no significant difference for ketoacids. In 10 animals, plasma FFA concentrations were determined. In nine animals, the arterial concentration was higher suggesting a net uptake of FFA by the pregnant uterus. Lactate production by the uterus accounted for approximately 18% of uterine glucose uptake if glucose is assumed to be the only source of uterine lactate production. The mean glucose/oxygen quotient across the uterus corrected for lactate output was 0.92 ± 0.34. It is concluded that glucose is a major metabolic substrate for the near-term uterus in the pregnant guinea pig and can account for most of the uterine oxygen consumption.