Normal human diploid cells have a limited life span and undergo replicative senescence after various limited population doublings. Cells must pass the senescence barrier to become immortal. The exact mechanisms of immortalization are not clear, although inactivation of the RB pathway, and/or the p53 pathway and activation of telomerase has been shown to be necessary for immortalization of certain cell types with DNA viruses or hTERT. Methylation-associated inactivation of tumor suppressor genes plays an important role in tumor progression. To test if gene-specific methylation contributes to the immortalized and transformed phenotype, we analyzed the methylation status of 17 genes in normal cells immortalized with SV40, hTERT, Ad5, Ad12-SV40 or HPV-18. Some of these immortalized lines were progressively transformed and tumorigenic in nude mice. We observed gene-specific methylation in the in vitro immortalized and transformed cells. SV40 and HPV18 immortalization resulted in different methylation spectra. In SV40- and h-TERT-immortalized prostate epithelial cells, the most frequently methylated gene was RASSF1A, while in HPV18-immortalized cell lines, the RAR-beta2 gene was universally methylated. Immortalization with SV40 resulted in methylation of a greater number of genes than immortalization with HPV. Furthermore, in SV40-immortalized cell lines, methylation affected different genes in fibroblasts compared with epithelial cells, suggesting that different mechanisms may be used by SV40 to immortalize cell lines of different origins. In HPV18-immortalized and subsequently transformed cell lines, the most commonly methylated genes were hormone responsive genes, such as AR, ER-beta and RAR-beta2. In general, more genes were methylated in neoplastically-transformed cell lines than in only immortalized cell lines, indicating that accumulation of epigenetic abnormalities may contribute to oncogenesis.