The incidence of diabetes is high in cystic fibrosis (CF) and is an important cause of morbidity and mortality. Understanding the pathophysiology is imperative. Studies have documented increased endogenous (mostly hepatic) glucose production (HGP) but have not distinguished the relative contribution of gluconeogenesis (GNG). The purpose of this study was to quantitate GNG, to determine its contribution to high HGP, and to measure insulin's suppression of GNG. We recruited 31 adult CF subjects (age, 26.2+/-7.9 years; 12 female subjects) and quantified GNG by measuring the incorporation of H into the second and fifth carbons of glucose. Hepatic glucose production was measured using [6,6-H2]glucose. Protein breakdown was measured using [1-C]leucine. Data were compared with that from 11 healthy volunteers (age, 27.5+/-7.0 years) who underwent both GNG and clamp studies. Thirteen CF subjects and all controls had a hyperinsulinemic euglycemic clamp during measures of GNG. Other measures included glucose tolerance and glucagon and cortisol levels. Rate of GNG was higher in CF subjects than controls and comprised a greater percentage of fasting HGP (GNG as percent of HGP: CF=68%; controls=44%; P=0.034). Suppression of GNG by insulin was significantly lower in CF than in controls and was lower in CF subjects with abnormal glucose tolerance than in those with normal glucose tolerance. Gluconeogenesis correlated with protein breakdown. These studies suggest that high HGP in CF is mostly from elevated rates of GNG and that resistance to insulin's suppression of GNG may contribute to abnormal glucose tolerance in CF.