Opioid binding protein/cell adhesion molecule-like gene (OPCML), a recently identified tumor-suppressor, is frequently inactivated by allele loss and CpG island promoter methylation in epithelial ovarian cancer. Since elevated activation of the RAS signaling pathway, including overexpression of HER-2/neu and mutations of RAS and BRAF, is common in human ovarian carcinoma, we examined the cellular effect of oncogenic RAS on the expression status of OPCML in a genetically defined human ovarian cancer model. Our study revealed that RAS(V12)-mediated oncogenic transformation was accompanied by a concomitant loss of OPCML expression. Methylation-sensitive PCR analysis showed that the OPCML promoter was hypermethylated in RAS-transformed human ovarian epithelial cells (T29H) and that treatment with the DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine promoted demethylation of the OPCML promoter and restored OPCML expression in T29H cells. Furthermore, suppression of oncogenic RAS activity by stable siRNA specific for HRAS(V12) led to the demethylation and re-expression of OPCML in T29H cells, demonstrating that oncogenic RAS activity is directly responsible for the observed OPCML promoter hypermethylation and epigenetic gene silencing of OPCML. Taken together, our study suggests that elevation of the RAS signaling pathway may play an important role in epigenetic inactivation of OPCML in human epithelial ovarian cancer.