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AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma.METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSF1A.RASSF1A mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3.Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSF1A promoter were evaluated by methylation specific PCR (MS-PCR).RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSF1A mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939cell lines (x2 = 14.270, P= 0.001＜0.01) showed RASSF1A express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%.CONCLUSION: The data we present suggest that RASSF1A which we have been searching for at 3p21.3may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSF1A.