Altered growth and differentiation and a highly abnormal karyotype are generally believed to be indicators for tumorigenic conversion of human cells. Inactivation of TP53 is supposedly one possible mechanism for accelerated genetic aberrations via reduced control of the genetic integrity. To examine the significance of this functional relationship, we investigated the long-term development of the spontaneously immortalized human skin keratinocyte line HaCaT, carrying UV-specific mutations in both alleles of the TP53 tumor suppressor gene. During > 300 passages, proliferation, clonogenicity, and serum-independent growth potential increased. In addition, HaCaT cells gained anchorage independence and at late passages showed reduced differentiation. Karyotypic analysis up to passage 225 revealed a high frequency of translocations and deletions, with a particular increase during passages 30 and 50. Nevertheless, the HaCaT cells remained nontumorigenic when injected subcutaneously, and noninvasive in surface transplants in nude mice. By comparative genomic hybridization, we confirmed the karyotypically identified phase of increased chromosomal aberrations between passages 30 and 50. However, before and thereafter, the CGH profiles of the individual chromosomes were largely unchanged, demonstrating that those translocations-also maintained in later passages-did not cause a gross chromosomal imbalance. Thus, our data suggest that multiple changes often correlated with a "transformed phenotype," including extensive karyotypic alterations and mutational inactivation of TP53, are well compatible with a nontumorigenic phenotype of the HaCaT cells, and that preserved chromosomal balance may be crucial for this stability during long-term propagation.