Atractyloside inhibited gluconeogenesis from dihydroxyacetone in hepatocytes from fasted rats and increased lactate synthesis. In the presence of atractyloside, lactate/pyruvate and beta-hydroxybutyrate/aceto-acetate ratios were increased and the accumulation of Fru-2,6-P2 was prevented. In the absence of atractyloside, gluconeogenesis from dihydroxyacetone was stimulated by dibutyryl-cAMP and, to a much lesser extent, by norepinephrine and vasopressin. Omission of Ca2+ increased the stimulation by norepinephrine but prevented that by vasopressin. High concentrations (greater than or equal to 40 microM) of atractyloside abolished the stimulation of gluconeogenesis by dibutyryl-cAMP but not that by norepinephrine or vasopressin. Exogenous Ca2+ was not required for hormonal stimulation in the presence of atractyloside. The stimulation by norepinephrine was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N-tetraacetic acid or prazosin but not by propranolol. Atractyloside caused decreases of all glycolytic intermediates and an activation of pyruvate kinase. Norepinephrine partially reversed these effects. The mitochondrial and cytosolic ATP/ADP ratios were determined by digitonin fractionation of hepatocytes. Norepinephrine or vasopressin increased the cytosolic ATP/ADP in the presence of atractyloside. We suggest that the increased availability of cytosolic ATP could be responsible for the stimulation of gluconeogenesis by these hormones.