Parenchymal heterogeneity in lactate disposal by perivenous and periportal hepatocytes is believed to be an important factor affecting the overall lactate metabolism by the liver. The aim of this work was to investigate the possibility of the existence of a different role of both acinar zones 1 and 3 in lactate secretion into bile. The effect of insulin and glucose load was also studied using isolated in situ rat liver preparations. Perfusions with erythrocyte-free Krebs-Henseleit solutions were carried out in intact livers and after restricted damage of zone 1 or zone 3 by digitonin administration as a bolus through the portal or the hepatic vein, respectively. In intact livers lactate concentrations in bile were similar to those found in the perfusate. In both compartments lactate concentrations were observed to increase over 90 min of perfusion. During this time, bile lactate output increased from 5 to 8 nmol/min per g liver with no significant effect on bile flow. Replacement of perfusate by a fresh lactate-free one at 60 min failed to induce any reduction in lactate concentration in bile samples collected during the following 30 min which suggests the absence of easy equilibration of biliary lactate with the sinusoidal compartment. Insulin administration (bolus: 100 mU/100 g body weight, plus portal infusion: 5 mU/min per 100 g body weight) was found to markedly enhance bile lactate concentrations (+110%) and output (+139%). On the contrary, glucose load was found to have no effect on lactate output into bile. No significant difference in the increase in bile lactate output was observed during 90 min perfusion with either 0, 5, 10, 15, 25 or 35 mM initial glucose concentrations. After restricted damage of acinar zone 1 or 3, insulin-induced bile lactate secretion was significantly reduced. This effect was not different regardless the damaged acinar zone. In summary these results suggest that insulin plays an important role in the control of the output of lactate into bile and that the existence of acinar heterogeneity in this function seems unlikely. Moreover the quantitative contribution of bile lactate to overall lactate handling by the liver and to bile formation seems very low.