Glucagon and L-epinephrine stimulate gluconeogenesis from 20 mM L-lactate, the effect being about 3 times greater in liver cells from fed rats than in those from fasted rats. The rate of pyruvate kinase flux was estimated to be less than 10% of the rate of gluconeogenesis from lactate in hepatocytes from fasted rats, and neither glucagon nor epinephrine lowered the absolute rate significantly. In hepatocytes from fed rats, however, the rate of pyruvate kinase was nearly one-half that of gluconeogenesis. Glucagon caused a marked depression of pyruvate kinase flux, with 1 muM glucagon lowering the rate to nearly the level found in cells from fasted rats Epinephrine at concentrations from 10(-8) to 10(-6) M actually increased pyruvate kinase flux during gluconeogenesis from lactate in cells from fed rats. These results are in accord with the view that the effects of glucagon and epinephrine on gluconeogenesis are not identical.