Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities. (HEPATOLOGY 1991;13:73–82).