Liver biopsy specimens with or without chronic liver diseases were examined immunohistochemically to determine the distribution of enolase isozymes (alpha, beta, and gamma). In normal liver, alpha-enolase was positively stained in almost all hepatocytes and bile duct cells. beta- and gamma-enolases were localized in hepatocytes and bile duct cells, respectively. Electron microscopic studies revealed that Kupffer cells and sinusoidal endothelial cells had both alpha- and gamma-enolases. In chronic active hepatitis and cirrhosis, proliferated biliary ductular cells had both alpha- and gamma-enolases, but did not express beta-enolase. This is almost the same localization pattern of enolase isozymes as in preexisting bile duct cells. gamma-Enolase was detected in some hepatocytes in eight of 12 cases with chronic active hepatitis and six of 12 cases with cirrhosis. These hepatocytes were small, showed a cobblestone pattern, and binucleate cells were frequent. On the other hand, rosette-formed hepatocytes adjacent to a regenerating bulging lobule were not stained for gamma-enolase. These results suggest that regenerating hepatocytes have gamma-enolase and that, with maturation, hepatocytes lose it.