The mechanisms responsible for the stimulation by phenylephrine of gluconeogenesis from dihydroxyacetone and glycerol were studied in perifused rat hepatocytes. The stimulation by phenylephrine of glucose formation from dihydroxyacetone was biphasic. Transient stimulation of about 25% after 3 min was followed by a stable stimulation of about 15% 7 min later. Concomitantly there was a transient inhibition by phenylephrine of pyruvate kinase flux during the first few minutes followed by a more stable inhibition after about 10 min. The stable inhibition could quantitatively account for the stable stimulation of gluconeogenesis by the hormone when dihydroxyacetone was the gluconeogenic substrate. The effects of phenylephrine were independent of cAMP. With the reduced gluconeogenic substrate, glycerol, a transient aminooxyacetate-sensitive stimulation of gluconeogenesis occurs, followed by a stable stimulation of smaller magnitude [Leverve, X. M., Groen, A. K., Verhoeven, A. J. and Tager, J. M. (1985) FEBS Lett. 181, 43-46]. This transient phase of stimulation was abolished by addition of low concentrations of pyruvate, in accordance with our previous proposal that phenylephrine transiently accelerates the mitochondrial oxidation of cytosolic NADH via the malate/aspartate shuttle. The transient stimulation of gluconeogenesis from glycerol was accompanied by a transient increase in the cytosolic/mitochondrial aspartate concentration gradient. Phenylephrine caused a transient stimulation of mitochondrial aspartate efflux in two other systems dependent on the operation of the malate/aspartate shuttle, i.e. gluconeogenesis from lactate and the oxidation of ethanol. It is proposed that addition of phenylephrine to hepatocytes brings about a transient increase in the mitochondrial membrane potential and hence an increased rate of transport of aspartate from the mitochondria to the cytosol.