Gluconeogenesis from lactate and pyruvate and associated parameters were investigated in the bivascularly and hemoglobin-free perfused rat liver. The substrates were infused either via the portal vein (anterograde perfusion mode), via the hepatic vein (retrograde mode) or via the hepatic artery (anterograde and retrograde modes). The rates of lactate and pyruvate infusion were 10.3 and 3.5 mumol min-1 g-1, respectively. The metabolic rates measured when the substrates were infused into the hepatic artery were referred to the cellular spaces accessible in each perfusion mode. The following results were obtained when the substrates were infused into the hepatic artery: (1) gluconeogenesis from lactate was equal to 2.08 +/- 0.2 mumol min-1 ml-1 in the retrograde mode and 1.33 +/- 0.08 mumol min-1 ml-1 in the anterograde mode (P = 0.019); (2) gluconeogenesis from pyruvate was equal to 0.66 +/- 0.11 mumol min-1 ml-1 in the retrograde mode and 0.7 +/- 0.11 mumol min-1 ml-1 in the anterograde mode (P = 0.78); (3) oxygen uptake increase with lactate was 1.75 +/- 0.14 mumol min-1 ml-1 in the retrograde mode and 1.05 +/- 0.07 mumol min-1 ml-1 in the anterograde mode (P = 0.002); (4) oxygen uptake increase with pyruvate was equal to 0.59 mumol min-1 ml-1 in the retrograde mode and 0.57 +/- 0.05 mumol min-1 ml-1 in the anterograde mode (P = 0.73); (5) pyruvate production from lactate was 0.28 +/- 0.06 mumol min-1 ml-1 in the retrograde mode and 0.39 +/- 0.05 mumol min-1 ml-1 in the anterograde mode (P = 0.28); (6) lactate production from pyruvate was equal to 0.52 +/- 0.05 mumol min-1 ml-1 in the retrograde mode and 0.99 +/- 0.08 mumol min-1 ml-1 in the anterograde mode (P < 0.001). Since only periportal cells are supplied with substrates when they are infused via the hepatic artery in retrograde perfusion, these results allow the conclusion that gluconeogenesis from lactate predominates in periportal hepatocytes. When pyruvate is the sole substrate, however, gluconeogenesis in periportal and perivenous cells presents no difference.