Objective:To examine the expression and promoter CpG island methylation of glutathione S-transferase M3 (GSTM3) gene in clear cell renal cell carcinoma (ccRCC),and to evaluate the relationship of expression,methylation of GSTM3 gene with the metastasis and oncogenesis of ccRCC.Methods:Using semi-quantitative RT-PCR technique,we examined GSTM3 expression in surgical specimens of 24 primary ccRCCs and adjacent non-malignant tissues,14 metastatic ccRCCs and 2 ccRCC cell lines (RCC05-TXJ,RCC05-ZTJ) with different metastatic potentials.RCC05-TXJ cells were cultured in RPMI 1640 medium and treated with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR).Semi-quantitative RT-PCR was used to examine the expression of GSTM3 in response to 5-Aza-CdR treatment.Nested bisulfite sequencing PCR and DNA sequencing were used to analyze different methylation locuses in GSTM3 gene promoter in 10 primary ccRCCs and adjacent non-malignant tissues.We also examined the methylation level in 10 primary ccRCCs and the corresponding non-malignant tissues as well as 8 metastatic tissues by nested methylation-specific PCR.Results:Expression of GSTM3 gene in metastatic ccRCC cells (RCC05-TXJ) was lower than that in the non-metastatic ccRCC cells (RCC05-ZYJ).Down-regulation of GSTM3 gene expression was found in 17 of the 24 primary ccRCCs as compared with the non-malignant tissue.Expression of GSTM3 in the metastatic ccRCCs was lower than that in primary ccRCCs.5-Aza-CdR treatment increased GSTM3 expression in RCC05-ZYJ．Methylation in GSTM3 promoter was found in 4 of 10 ccRCC tissues,2 of 10 adjacent tissues,and 1 of 8 metastatic tissues.No significant difference was found between ccRCC and adjacent tissues (P=0.628) or ccRCC and metastatic disease (P=0.314) due to limit number of cases.Conclusion:Our findings support that promoter aberrant methylation is one of the major mechanisms of GSTM3 gene down-regulation in ccRCC.The preliminary identification of GSTM3 promoter methylation sites may provide a basis for further study.