Although gallbladder carcinoma is the most common malignancy of the extrahepatic biliary tract, patients with this disease have a very poor prognosis. Previous investigations have documented the presence of estrogen receptors (ER) in gallbladder cancers for possible therapeutic benefit; however, the methods used for detection were largely imprecise, which led to a wide range of results. In light of these unclear results obtained with inferior techniques, we have attempted to determine more accurately the prevalence of ER positivity in gallbladder cancer using an improved, more specific immunohistochemical assay (IHA) for estrogen receptors. The IHA, which used a pronase pretreatment and the H222 monoclonal antibody, was performed on archival tumor specimens of 25 patients. There were no strongly or moderately positive specimens. Three specimens stained weakly positive, and 22 specimens (88%) were negative. The three specimens that were weakly positive showed neither statistical significance nor any identifiable trends with respect to patient sex, tumor stage, or histologic type. We conclude that, contrary to previous reports, weak estrogen receptor staining occurs in a very small percentage of gallbladder carcinomas. Furthermore, although the minimum concentration of ER required for potential therapeutic benefit is unclear, the addition of hormonal therapy in patients with gallbladder cancer is not likely to alter the outcome of this malignancy.