The present study was conducted to examine the effect of a single bout of exercise (rodent treadmill, 60 min at 26 m/min, 0% grade) on the gluconeogenic activity of periportal hepatocytes (PP-H) and perivenous hepatocytes (PV-H) in fasted (18 h) rats. Isolated PP-H and PV-H, obtained by selective destruction following liver perfusion with digitonin and collagenase, were incubated with saturating concentrations of alanine (Ala; 20 mM) or a mixture of lactate and pyruvate (Lac+Pyr; 20:2 mM) to determine the glucose production flux (J_glucose) in the incubation medium. Results show that, in the resting conditions, J_glucose from all exogenous substrates was significantly higher (P < 0.01) in PP-H than in PV-H. Exercise, compared with rest, resulted in a higher J_glucose (P < 0.01) from Lac+Pyr substrate in the PV-H but not in the PP-H, resulting in the disappearance of the difference in J_glucose between PP-H and PV-H. Exercise, compared with rest, led to a higher J_glucose (P < 0.01) from Ala substrate in both PP-H and PV-H. However, the exercise-induced increase in J_glucose (gluconeogenic activity) from Ala substrate was higher in PV-H than in PP-H, resulting, as from Lac+Pyr substrate, in the disappearance (P > 0.05) of the difference of J_glucose between PP-H and PV-H. It is concluded that exercise differentially stimulates the gluconeogenic activity of PV-H to a larger extent than PP-H, indicative of a heterogenous metabolic response of hepatocytes to exercise.