Widespread use of liver transplantation in the treatment of hepatic diseases is restricted by the limited availability of donated organs. One potential solution to this problem would be isolation and propagation of liver progenitor cells or stem cells. Here, we report on the isolation of a novel progenitor cell population from unmanipulated (that is, no prior exposure to chemicals and no injury) adult rat liver. Rat liver cells were cultured following a protocol developed in our laboratory to generate a unique progenitor cell population called liver-derived progenitor cells (LDPCs). LDPCs were analyzed by fluorescence-activated cell sorting, real-time polymerase chain reaction (RT-PCR), immunostaining and microarray gene expression. LDPCs were also differentiated into hepatocytes and biliary epithelium in vitro and examined for mature hepatic markers and urea and albumin production. These analyses showed that, LDPCs expressed stem cell markers such as cluster domain (CD)45, CD34, c-kit, and Thy 1, similar to hematopoietic stem cells, as well as endodermal/hepatic markers such as hepatocyte nuclear factor (HNF)3β, hematopoietically-expressed homeobox gene-1, c-met, and transthyretin. LDPCs were negative for OV-6, cytokeratins (CKs), albumin, and HNF1α. The microarray gene expression profile demonstrated that they showed some similarities to known liver progenitor/stem cells such as oval cells. In addition, LDPCs differentiated into functional hepatocytes in vitro as shown by albumin expression and urea production. In conclusion, LDPCs are a population of unique liver progenitors that can be generated from unmanipulated adult liver, which makes them potentially useful for clinical applications, especially for cell transplantation in the treatment of liver diseases. Liver Transpl 14:333–345, 2008. © 2008 AASLD.