A potential role for muscle in glucose homeostasis was recently suggested based on characterization of extrahepatic and extrarenal glucose-6-phosphatase (glucose-6-phosphatase-beta). To study the role of extrahepatic tissue in glucose homeostasis during fasting glucose kinetics were studied in two patients with a deficient hepatic and renal glycogenolysis and/or gluconeogenesis. Endogenous glucose production (EGP), glycogenolysis (GGL), and gluconeogenesis (GNG) were quantified with stable isotopes in a patient with glycogen storage disease type 1a (GSD-1a) and a patient with fructose-1,6-bisphosphatase (FBPase) deficiency. The [6,6-H-2(2)]glucose dilution method in combination with the deuterated water method was used during individualized fasting tests. Both patients became hypoglycemic after 2.5 and 14.5 h fasting, respectively. At that time, the patient with GSD-1a had EGP 3.84 mu mol/kg per min (30% of normal EGP after an overnight fast), GGL 3.09 mu mol/kg per min, and GNG 0.75 mu mol/kg per min. The patient with FBPase deficiency had EGP 8.53 mu mol/kg per min (62% of normal EGP after an overnight fast), GGL 6.89 mu mol/kg per min GGL, and GNG 1.64 mu mol/kg per min. EGP was severely hampered in both patients, resulting in hypoglycemia. However, despite defective hepatic and renal GNG in both disorders and defective hepatic GGL in GSD-1a, both patients were still able to produce glucose via both pathways. As all necessary enzymes of these pathways have now been functionally detected in muscle, a contribution of muscle to EGP during fasting via both GGL as well as GNG is suggested.