Adult rat liver contains a minor population of hepatocytes called small hepatocytes (SHs) that are smaller in size and show a higher replicative potential than conventional parenchymal hepatocytes (PHs). However, SHs have been hitherto characterized using a "SH-fraction" that was contaminated with PHs. In the present study, we isolated a PH-free SH-fraction from the adult rat liver using fluorescence-activated cell sorter combined with centrifugal elutriation and characterized the hepatocytes in the fraction. These hepatocytes were designated R3Hs in this study. R3Hs were mononuclear and of lower ploidy. They expressed at high levels genes of Cdc2, connexin 26, hydroxysteroid sulfotransferase, pancreatic secretory trypsin inhibitor, and prostaglandin E2 receptor EP3 subtype. We conclude that SHs dominate the periportal zone in the adult liver, because mRNA or proteins of these genes were exclusively expressed by periportal hepatocytes.