To investigate whether protein, carbohydrate, and fat metabolism was normalized in insulin-treated gestational diabetes mellitus (GDM), eight Hispanic women with GDM and eight healthy controls were studied at 32-36 wk of gestation and 6 wk postpartum. Net substrate utilization was measured using room respiration calorimetry. Exogenous substrate oxidation was determined by 13C recovered in breath CO2 from 13C-labeled leucine, glucose, and Hiolein. Women with GDM had higher 24-h oxygen consumption, carbon dioxide production, total energy expenditure, and basal metabolic rates than controls due to larger body mass. Adjusted for weight or fat-free mass, total energy expenditure, basal metabolic rate, and basal and 24-h whole body net protein, carbohydrate, and fat utilization did not differ between insulin-treated GDM subjects and controls in pregnancy or postpartum. Oxidation of [13C]leucine and [13C]glucose did not differ by group or pregnancy status. Recovery of exogenously administered [13C]Hiolein, a biosynthetic triglyceride, as breath 13CO2 was significantly lower in the GDM group antepartum and postpartum (P = 0.02), indicating lower oxidation of exogenous triglycerides in GDM.