In isolated perfused rat livers, infusion of phorbol 12-myristate 13-acetate (PMA) (150 nM) resulted in a 3-fold stimulation of the rate of glucose production. This response was maximal at a perfusate PMA concentration of 150 nM, and was significantly diminished at higher concentrations of PMA (e.g. 300 nM). Stimulation of glycogenolysis by PMA was greatly decreased in livers perfused with Ca2+-free medium. PMA infusion into livers perfused in the absence of Ca2+ did not result in Ca2+ efflux from the livers. Additionally, in hepatocytes isolated from livers of fed rats, neither PMA nor 1-oleoyl-2-acetyl-rac-glycerol stimulated the rate of glucose production. Although indomethacin has been demonstrated to block PMA-stimulated hepatic glycogenolysis [Garcia-Sainz & Hernandez-Sotomayor (1985) Biochem. Biophys. Res. Commun. 132, 204-209], infusion of PMA into perfused rat livers did not alter the rates of production of either prostaglandin E2 or 6-oxo-prostaglandin F1 alpha in the livers. These data, along with the observed increases in the perfusion pressure and decrease in O2 consumption in isolated perfused livers suggest that phorbol-ester-stimulated glycogenolysis is not a consequence of a direct effect of phorbol ester on liver parenchymal cells.