The temperature-sensitive mutant of the SV40 virus large T antigen (TAg) tsA58 is frequently employed for the conditional immortalization of primary cells. By increasing the temperature to 39 degreesC, the activity of the mutant TAg is reduced and the status of such cells may then resemble more closely that of primary cells. As an alternative, we used a novel immortalization vector with a tetracycline-regulated expression of the wild-type TAg. This enabled us to investigate the effects of the immortalizing gene expression and of temperature shifts independently of each other. Even for wild-type TAg-derived cell lines the elevated temperatures led to various clone-dependent phenotypes. This suggests that in freshly established cell lines temperature-sensitive growth phenotypes can arise spontaneously and independently of a temperature-sensitive immortalizing gene. Similar effects were observed with spontaneously immortalized cells. On the other hand, not all of the ts-TAg-derived cell lines were proliferation arrested at the non-permissive temperature. Therefore, the assumption that temperature-sensitive growth is solely due to the ts-TAg must be verified for each ts-TAg-derived cell line individually. This complexity could be avoided by using the autoregulatory immortalization vector expressing the wild-type TAg. (C) 2004 Elsevier Inc. All rights reserved.