Pretreatment with retrorsine crosslinks host hepatocyte DNA and prevents proliferation after partial hepatectomy (PH), allowing selective expansion of transplanted progenitors. Shortcomings are length of protocol and carcinogenicity of retrorsine. This report describes a rapid liver repopulation protocol using mitomycin C (MMC) to block proliferation of rat hepatocytes in response to PH. One week post-MMC treatment, dipeptidyl peptidase IV negative host rats were given a PH followed by injection of late gestation, newborn, or adult total liver isolates from dipeptidyl peptidase IV positive rats. For allogeneic transplantation, host rats received injections of anti-CD3 antibody before and after PH. Host liver staining 2 to 9 weeks posttransplantation revealed well-defined donor hepatocyte colonies with strong canalicular dipeptidyl peptidase IV activity. At the same cell dose, fetal and newborn isolates produced more colonies than adult liver isolates. Hepatocyte colonies also coexpressed marker proteins characteristic of adult hepatocytes and showed polarized localization of plasma membrane proteins. Host livers contained large clusters of sinusoids lined by dipeptidyl peptidase IV positive endothelial cells coexpressing the endothelial cell marker, RECA-1, but lacked the canalicular marker leucine aminopeptidase. Colonies containing donor hepatocytes, endothelial cells, and bile ducts were also observed. Similar levels of engraftment and expansion were achieved with allogeneic liver cell isolates by using anti-CD3 antibody treatment. The MMC transplantation model provides a rapid method for engraftment and expansion of hepatocytes, endothelial cells, and cholangiocytes and should be applicable to investigations centering on the role of endothelial cells in liver regeneration and the identification and characterization of putative endothelial, hepatocyte, and cholangiocyte progenitors.