BACKGROUND: During pregnancy, metabolic adaptation takes place in the mother to provide for the supply of substrates to the growing fetus. OBJECTIVE: To determine rates and endocrine regulation of lipolysis and glucose production (GPR) in late pregnancy. DESIGN: Energy substrate production was measured in healthy pregnant women by use of stable isotope-labelled compounds. SETTING: University Hospital, Uppsala, Sweden. SAMPLE: Eight healthy non-obese, non-smoking women with normal pregnancies were studied at 33-36 weeks of gestation after an overnight (12-14 hours) fast. METHODS: Rates of glycerol and glucose production were analysed by gas chromatography/mass spectrometry following constant rate infusion of [1,1,2,3,3-(2)H(5)]-glycerol and [6,6-(2)H(2)]-glucose. MAIN OUTCOME MEASURE: Glycerol and glucose production in the third trimester. RESULTS: The mean rate of glycerol production, reflecting lipolysis, was 3.06 (0.66) and the mean GPR was 13.2 (1.5) micromol kg(-1) minute(-1) [2.38 (0.27) mg kg(-1) minute(-1)]. There was a correlation between rate of glycerol production and GPR (r = 0.75, P = 0.033). Fasting insulin levels correlated inversely with both the rate of glycerol production (r = -0.85, P = 0.008) and GPR (r = -0.78, P= 0.021). CONCLUSIONS: Our results show that lipolysis is markedly increased during late pregnancy compared with reported data for non-pregnant women. The data also confirm the occurrence of an increased GPR in pregnant women. The finding of a correlation between rate of glycerol production and GPR corroborates the view that lipolysis promotes gluconeogenesis. Although late gestation is associated with insulin resistance, the results show that insulin plays a regulatory role both in lipolysis and glucose production.