An extensive bile ductular cell hyperplasia with the formation of well-differentiated bile ductules is the most prominent feature of rat liver at 6 to 15 weeks after bile duct ligation. We have improved our previous cell isolation procedure and are now routinely able to obtain from such livers high yields of viable bile ductular epithelial cells. These cells were characterized with respect to their specific activities of gamma-glutamyl transpeptidase and beta-glucuronidase and of select Phase I and Phase II enzymes of biotransformation. At the time of their isolation, only a very small number of the bile ductular epithelial cells were observed to be in DNA synthesis. In addition, in histological sections prepared from intact hyperplastic bile ductular tissue isolates, only the bile ductular epithelial cells exhibited histochemical staining for gamma-glutamyl transpeptidase activity. Typically, greater than 95% of the cells isolated from this tissue were also found to be histochemically positive for gamma-glutamyl transpeptidase activity, and no hepatocytes were seen contaminating this cell population. Biochemically, the isolated bile ductular cells exhibited a gamma-glutamyl transpeptidase specific activity that was 100 times higher than that of hepatocytes isolated at the same time from the bile duct-ligated rats and more than 300 times higher than the specific activity of the enzyme of freshly isolated normal rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)