To obtain hepatocyte subpopulations of defined intralobular regional origin, rat livers were perfusion-labeled with fluorescein diacetate. A high concentration of fluorescein diacetate solution was prepared with the aid of porcine serum albumin and pulse-infused into the rat liver in situ in an antero- or a retrograde mode. Fluorescein diacetate was rapidly and reversibly taken up by hepatocytes of afferent regions and migrated “chromatographically” along the sinusoid while undergoing very rapid hydrolysis to fluorescein. The migration rate was accelerated by higher albumin concentrations in the infusate and perfusate and by higher flow rates. Flrorescence photomicrographs showed a distinct labeling of afferent regions which appeared as the brightest labeled population in a flow cytometry plot of frequency vs log (fluorescence). The cytometry plot showed unlabeled hepatocytes as a major broad peak above autofluorescence and fluorescence-labeled cells as a plateau above the peak. The centrilobular hepatocytes sorted in a fluorescence-activated cell sorter contained about twofold as much activity for paraoxonase, paraoxon deethylase, and fluorescein diacetate hydrolase as the periportal hepatocytes of the liver from both control and DDE-treated rats. DDE-treated rat livers had about 15-fold higher paraoxon deethylase activity than controls while paraoxonase and fluorescein diacetate hydrolase were elevated about 1.5 times.