Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10(-5) M) as alpha-adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol-3-phosphate dehydrogenase and glycerol kinase.