We have previously achieved a high level of long-term liver replacement by transplanting freshly isolated embryonic day (ED) 14 rat fetal liver stem/progenitor cells (FLSPCs). However, for most clinical applications, it will be necessary to use cryopreserved cells that can effectively repopulate the host organ. In the present study, we report the growth and gene expression properties in culture of rat FLSPCs cryopreserved for up to 20 months and the ability of cryopreserved FLSPCs to repopulate the normal adult rat liver. After thawing and placement in culture, cryopreserved FLSPCs exhibited a high proliferation rate: 49.7% Ki-67-positive on day 1 and 34.7% Ki-67-positive on day 5. The majority of cells were also positive for both α-fetoprotein and cytokeratin-19 (potentially bipotent) on day 5. More than 80% of cultured cells expressed albumin, the asialoglycoprotein receptor, and UDP-glucuronosyltransferase (unique hepatocyte-specific functions). Expression of glucose-6-phosphatase, carbamyl phosphate synthetase 1, hepatocyte nuclear factor 4α, tyrosine aminotransferase, and oncostatin M receptor mRNAs was initially negative, but all were expressed on day 5 in culture. After transplantation into the normal adult rat liver, cryopreserved FLSPCs proliferated continuously, regenerated both hepatocytes and bile ducts, and produced up to 15.1% (mean, 12.0% ± 2.0%) replacement of total liver mass at 6 months after cell transplantation. These results were obtained in a normal liver background under nonselective conditions. This study is the first to show a high level of long-term liver replacement with cryopreserved fetal liver cells, an essential requirement for future clinical applications.