The uptake of substrates by the pregnant uterus defines the "diet" of the uterus and conceptus. In order to determine the uterine substrate uptake, catheters were placed in the femoral artery and uterine veins of guinea pigs at 40-49 days gestation. After at least 2 days recovery from perioperative stress, systemic arterial and uterine venous concentrations of oxygen, glucose, lactate, acetoacetate, beta-hydroxybutyrate, acetate, and free fatty acids were measured and metabolic quotients were calculated. The glucose/O2 quotient was 1.22 +/- 0.07 (mean +/- SEM), the lactate/O2 quotient was -0.40 +/- 0.09, the acetoacetate/O2 quotient was 0.03 +/- 0.02, the beta-hydroxybutyrate/O2 quotient was 0.01 +/- 0.006, the acetate/O2 quotient was 0.03 +/- 0.01, and the free fatty acid/O2 quotient was 0.24 +/- 0.21. Among the substrates measured, glucose appears to be the major metabolic fuel of the pregnant guinea pig uterus, but does not account for all of the oxygen consumed by the uterus if adjusted for lactate production, since lactate is produced within the gravid uterus in large quantities. Acetoacetate and beta-hydroxybutyrate are utilized in negligible amounts.