Hepatic oval cells (HOC) are a small subpopulation of cells found in the liver when hepatocyte proliferation is inhibited and followed by some type of hepatic injury. HOC can be induced to proliferate using a 2-acetylaminofluorene (2-AAF)/hepatic injury (i.e., CCl 4 , partial hepatectomy [PHx]) protocol. These cells are believed to be bipotential, i.e., able to differentiate into hepatocytes or bile ductular cells. In the past, isolation of highly enriched populations of these cells has been difficult. Thy-1 is a cell surface marker used in conjunction with CD34 and lineage-specific markers to identify hematopoietic stem cells. Thy-1 antigen is not normally expressed in adult liver, but is expressed in fetal liver, presumably on the hematopoietic cells. We report herein that HOC express high levels of Thy-1. Immunohistochemistry revealed that the cells expressing Thy-1 were indeed oval cells, because they also expressed β£-fetoprotein (AFP), β₯-glutamyl transpeptidase (GGT), cytokeratin 19 (CK-19), OC.2, and OV-6, all known markers for oval cell identification. In addition, the Thy-1 Ψ cells were negative for desmin, a marker specific for Ito cells. Using Thy-1 antibody as a new marker for the identification of oval cells, a highly enriched population was obtained. Using flow cytometric methods, we isolated a 95% to 97% pure Thy-1 Ψ oval cell population. Our results indicate that cell sorting using Thy-1 could be an attractive tool for future studies, which would facilitate both in vivo and in vitro studies of HOC. (HEPATOLOGY 1998;27:433-445.) Adequate data have been gathered that show that oval cells exist in the treated liver, but their place of origin and their role in liver development, regeneration, and carcinogenesis remains enigmatic. Oval cells increase in numbers when hepatocyte proliferation is suppressed. 2-Acetylaminofluorene (2-AAF) given before and during hepatic injury by a partial hepatectomy of two thirds (PHx) results in suppres-