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Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.