Protein gene product 9.5 (PGP9.5) is a neurospecific peptide that removes ubiquitin from ubiquitinated proteins and prevents them being targeted for degradation by proteosomes. Its expression is a potential marker of non-small lung cancer, invasive colorectal cancer and esophageal squamous cell carcinoma. Gallbladder (GB) cancer is the most common malignant tumor of the biliary tract and is usually associated with gallstone disease, a late diagnosis, unsatisfactory treatment and a poor prognosis. To understand the role of PGP9.5 in GB cancer, we examined the methylation status of its promoter and its expression in surgical biopsy samples. Formalin-fixed, paraffin-embedded tumors and non-neoplastic GB tissues (22 carcinomas, eight adenomas, 26 normal epithelia) were collected from patients who had undergone surgical resection. The methylation status of the promoter region of the PGP9.5 gene was determined by methylation-specific polymerase chain reaction, and the expression of PGP9.5 was examined by immunohistochemistry using tissue microarrays. PGP9.5 promoter was methylated in 84.6% (22/26) of normal GB epithelium, 37.5% (3/8) of adenomas and 27.2% (6/22) of carcinomas. Most tumors with an unmethylated promoter exhibited positive staining for PGP9.5 in epithelial and neoplastic cells, but no PGP9.5 expression was observed in normal epithelia or in tumor tissues with a methylated promoter. No correlation was found between promoter hypomethylation of PGP9.5 and clinicopathological findings (i.e. age, sex, histological type or grade, N-status, invasion depth or tumor stage) whereas PGP9.5 hypomethylation was found to be inversely correlated with the presence of a gallstone (P = 0.015). These results suggest that PGP9.5 promoter hypomethylation could be a reliable marker for GB cancer and that DNA hypomethylation might play an important role in re-expression of the PGP9.5 gene in GB cancer.