Based on previous studies, a minimal set of genetic alterations that is required to convert normal human fibroblasts into cancer cells has been defined. Essential roles for telomere maintenance and alterations in phosphatase 2A activity were inferred from experiments in which tumorigenicity was tested by injecting cells under the skin of immunodeficient mice. However, in the present experiments, the combination of SV40 large T antigen and activated Ras, without hTERT or SV40 small t antigen, was sufficient to convert nine different primary human fibroblast cell strains to a fully malignant state. The malignant behavior of the cells was demonstrated by growth of the cells into invasive tumors when the cells were injected beneath the kidney capsule of immunodeficient mice. Lung metastases and circulating tumor cells were also detected. These tumors were not immortal; cells entered crisis, from which they could be rescued by expression of hTERT. However, the same cell populations were not tumorigenic when they were injected under the skin. In this site, tumorigenicity required the expression of hTERT and SV40 small t antigen as well as SV40 large T antigen and Ras. The cellular pathways targeted by SV40 large T antigen (p53 and pRb) and those targeted by activated Ras represent a minimal set of genetic alterations required for the conversion of normal human fibroblasts into cancer cells.