OBJECTIVE: To explore potential epigenetic biomarkers for toxic effects, tumor-related chemical prevention and biological monitor by a genome-wide screening for differential DNA methylation during human cell malignant transformation in vitro. METHODS: The two in vitro cell transformation models included B(a)P-induced human bronchial epithelial cell introduced by H-Ras (HBER) cell transformation and simian vacuolating virus 40 small T antigen induced (SV40 ST-induced) HBER cell transformation. Methylated genes were collected by methylated DNA immunoprecipitation and whole genome amplification (MeDIP-WGA) at three time points during cell transformation which represented different transformation stage. Then, CpG island microarray was used to screen differentially methylated genes. The mRNA levels of hypermethylated genes were also observed by RT-PCR. RESULTS: The CpG island microarray showed that the number of hypermethylated genes in HBER, HBERNT, HBERT cells were 733, 661 and 738 respectively.83 genes were hypermethylated in pre-transformed cell and transformed cell. Moreover, 25 of 83 genes were also hypermethylated in SV40 ST-transformed cell (HBERST). We further confirmed that the mRNA expression of six of these 25 genes, namely family with sequence similarity 178, member A (FAM178A), retinoic acid receptor responder (tazarotene induced) (RARRES1), ubiquitin specific peptidase 28 (USP28), Scm-like with four mbt domains 2 (SFMBT2), family with sequence similarity 59, member A (FAM59A) and nuclear receptor subfamily 4, group A, member 3 (NR4A3) were suppressed during B(a)P-induced transformation. CONCLUSION: The abnormal hypermethylation of specific genes was a common event in the two kinds of human cell transformation models, which shed light on the study for chemical exposure monitor and tumor-related epigenetic biomarkers.