After a short-term fast, lactating women have increased rates of glucose production but not gluconeogenesis (GNG) despite relative hypoinsulinemia. We explored the effects of non-insulin-dependent increase in glucose utilization and recombinant human growth hormone (rhGH) on glucose production, glycogenolysis, and GNG in both the fed and overnight-fasted condition. Six controls and 7 lactating women were studied twice, in random order, after 7 days of saline or rhGH. Glucose kinetics and GNG were measured using [U-(13)C]glucose mass isotopomer distribution analysis. The rhGH increased milk production in the lactating women and insulin-like growth factor (IGF) in both groups. Glycogenolysis and GNG were higher in fasting lactating women than controls after either saline or rhGH (P < .05). After rhGH administration, GNG remained higher (P < .02) in the lactating women than controls. Gluconeogenesis was not suppressed in either group during 5 hours of continuous meal ingestion, despite a 5-fold increase in plasma insulin. Lactating women had similar glucose but lower insulin and C-peptide concentrations than controls after both rhGH and saline treatment (P < .01), although rhGH decreased (P < .01) insulin sensitivity in both groups (P < .05). Gluconeogenesis is not affected by short-term increases in insulin and/or rhGH, which suggests a fundamental rethinking of the role of insulin in acutely regulating GNG.