We have described recently a human fibroblast cell line immortalized through ectopic telomerase expression (cen3tel), in which the extension of the life span was associated with the appearance of chromosomal aberrations and with the ability to grow in the absence of solid support. As reported in this article, on further propagation in culture, cen3tel cells became neoplastically transformed, being able to form tumors in nude mice. The analysis of the cells, during the gradual transition toward the tumorigenic phenotype, allowed us to trace cellular and molecular changes associated with different phases of transformation. At the stage in which they were able to grow in agar, cen3tel cells had lost contact growth inhibition but still retained the requirement of serum to proliferate and were not tumorigenic in immunocompromised mice. Moreover, they showed a down-regulation of the INK4A locus and were resistant to oncogenic Ras-induced senescence but still retained a functional p53. Subsequently, cen3tel cells became tumorigenic, lost p53 function because of a mutation in the DNA-binding motif, and overexpressed c-myc. Interestingly, tumorigenic cells did not carry activating mutations either in the ras proto-oncogenes (H-ras, N-ras, and K-ras) or in B-raf. Cen3tel cells gradually became hyperdiploid but did not display centrosome abnormalities. To our knowledge, cen3tel is the first telomerase immortalized fibroblast line, which became neoplastically transformed. In this system, we could associate a down-regulation of the INK4A locus with anchorage-independent growth and with resistance to Ras-induced senescence and link p53 mutations and c-myc overexpression with tumorigenicity.