Postsurgical recurrence at distant sites frequently occurs in pathological tumor stage 2 (pT2) carcinoma of the gallbladder even though the carcinoma is limited to the gallbladder wall. Little is known, however, about the molecular events leading to its development and progression. A large body of evidence suggests that cyclooxygenase-2 (COX-2) is up-regulated in carcinoma tissues and plays roles in promoting cell-proliferation, growth and metastasis of carcinoma cells. In the present study, immunohistochemistry was performed to determine the expression levels of COX-2 in the subserosal layer of 33 cases of pT2 gallbladder carcinoma in which curative resections had been performed and to determine the correlations of the expression levels of COX-2 with mode of recurrence and postsurgical survival. Immunostaining of COX-2 in the epithelia was recognized in more than 80% of normal epithelia, noncancerous pathological lesions of the gallbladder except for intestinal metaplasia and pT1–4 carcinoma specimens. Intense staining was observed in large percentages of hyperplastic lesions (65%), pT2 carcinoma specimens (76%) and pT3 and pT4 carcinoma specimens (64%) compared to the percentages of normal epithelia and other pathological lesions (0–25%). Intense staining was also observed in the adjacent stroma in pT2 carcinoma specimens (33%) and in those in pT3 and pT4 carcinoma specimens (43%) but only in small percentages of the stroma adjacent to normal epithelia and pathological lesions (0–8%). In situ hybridization confirmed the existence of COX-2 mRNA in both the cancerous epithelia and adjacent stroma of pT2-pT4 carcinomas. In 33 cases of pT2 carcinoma, distant recurrence, i.e., liver metastasis, was seen in 3 of 9 cases of pT2 carcinoma (33%, P<0.05) with intense stromal staining in the subserosal layer and in 1 of 24 cases (4%) without intense staining, whereas no significant correlation was found between parameters of pathological malignancies (histological grade, lymphatic permeation, venous permeation and lymph node metastasis) and the intensity of stromal staining in the subserosal layer. The postsurgical survival outcome was significantly poorer in the former than in the latter (p = 0.010). In pT2 gallbladder carcinoma, upregulation of COX-2 in the stroma adjacent to the cancerous epithelia in the subserosal layer correlates with the aggressiveness of the disease, such as the tendency to form distant recurrences. This phenotype may serve as a unique biological feature associated with the malignant behavior of pT2 gallbladder carcinoma. © 2002 Wiley-Liss, Inc.