A 'bottle-neck' for construction of autologous genetically engineered tumor vaccines and characterization of tumor antigens consists in the difficulty of establishing cell lines from human tumor material. We have constructed two retroviruses allowing transient expression of Simian virus 40 large T as an immortalizing agent. The first vector contains the genes for hygromycin and Herpes Simplex Virus thymidinkinase (TK), for positive and negative selection and the gene encoding large T. They are flanked by LoxP sites, the substrate of the bacteriophage recombinase Cre. The second retrovirus contains the genes for the Cre recombinase and puromycin as selection marker. By sequential infection of NIH3T3 cells with the two viruses, we have shown that the newly expressed large T gene can be deleted in a large proportion (> or =90%) of cells by site-specific recombination. Because the deletion included the TK gene, selection with gancyclovir against cells not having undergone recombination was possible. By infection with the large T retrovirus, cell lines could be easily established from mouse primary kidney cells, human fibroblasts, and cells derived from different surgical specimens of breast or colon cancer patients. One breast carcinoma cell line was further analyzed and shown to be of epithelial origin by characteristic markers (cytokeratins, mucin). This cell line grew continuously in culture for more than a year without any indication of a cell crisis. Infection with the cre-puro retrovirus and GCV selection resulted in complete excision of the large T gene as judged from antibody staining. Remarkably, these cells changed morphology and stopped proliferation comparable to the cells obtained from biopsy demonstrating the requirement of large T for growth. Therefore, this approach may facilitate molecular and cellular characterization of human tumors and other cell types where cell culturing is the limiting step, and gene therapy approaches involving autologous tumor cells.