Epigenetical inactivation of the RASSF1A tumor suppressor gene was frequently detected in cancer. However, the mechanisms of aberrant DNA methylation in the RASSF1A CpG island are unknown. In the present study, four Sp1 sites in the RASSF1A promoter were characterized; and the functional relationship between DNA methylation, histone modification, Sp1 binding and RASSF1A expression was examined in proliferating human mammary epithelial cells. With increasing passages of HMECs, the transcription of RASSF1A was dramatically silenced and this was associated with deacetylation and lysine 9 trimethylation of histone H3 and spreading DNA methylation from upstream and downstream into promoter. The RASSF1A CpG island in HMECs, which had overcome a stress-associated senescence barrier, was characterized by de novo DNA methylation and an elevated level of trimethylated histone H3 lysine 9. The binding of Sp1 to the RASSF1A promoter was impaired in these cells. The present data suggest that the chromatin inactivation occurs in the same time window as gene inactivation and may precede the de novo DNA methylation. Moreover, present results indicate that the Sp1 binding is mediated by chromatin state and not by DNA methylation. In summary, progressive histone inactivation, spreading of DNA methylation and inactivation of the Sp1 binding were observed in the RASSF1A promoter during senescence of HMECs and this system may serve as a model for the epigenetical inactivation of the tumor suppressor gene in carcinogenesis.