The objective of this study was to isolate hepatocytes of the proximal half (Zones 1 and 2) or distal half (Zones 2 and 3) of the liver acinus. The zonal origin of the isolated hepatocytes was recognized by: the presence in hepatocytes of a fluorescent marker, acridine orange, selectively delivered to either the proximal or the distal half of the acinus by in situ perfusion prior to cell isolation and the measurement of the induction of cytochrome P-450 by phenobarbital, an induction known to occur predominantly in the distal half of the acinus. Following the selective labeling of the acinus with acridine orange, livers were perfused with collagenase in either the portal to hepatic vein direction (anterograde) or in the retrograde direction. Hepatocytes isolated by either an anterograde or a retrograde perfusion were separated by centrifugation in a Percoll density gradient. This procedure isolated populations of proximal or distal hepatocytes, respectively, which were intact and 90% fluorescent. In an effort of assessing the heterogeneity of the separated proximal and distal hepatocytes, each population was further fractionated by centrifugal elutriation. This resulted in the arbitrary separation of proximal or distal hepatocytes into five fractions. Total cytochrome P-450 was determined spectrophotometrically in each of the fractions isolated from controls and after 3 days of the in vivo administration of phenobarbital. On the basis of the pattern of fluorescence in isolated hepatocytes and on the cytochrome P-450 inductive response to phenobarbital administration, it is proposed that: the anterograde or retrograde perfusion of the liver with collagenase separated hepatocytes predominantly of the proximal or distal half of the liver acinus, respectively and that hepatocytes of the distal half of the liver acinus responded to phenobarbital administration with the highest level of cytochrome P-450 induction, indicating that the isolated hepatocytes conserved the functional heterogeneity observed in vivo.