Aim: The significance of cell cycle proteins, p21 and p53, and cyclo-oxygenase-2 (COX2) is still controversial in gallbladder adenocarcinoma. Conflicting results exist with regard to the prognostic significance of p21 and p53 expression, and the strong association of gallbladder cancer with chronic inflammation makes it only natural that COX2 negativity is rarely encountered, even in non-neoplastic biliary epithelia. Methods: The authors explored whether the p21 and p53-labelling indices (LI) and strong cytoplasmic COX2 expression differ in the central parts (TC) and invasive fronts (IF) of gallbladder cancer. Paraffin-embedded whole tissue sections of 67 gallbladder cancers were immunohistochemically analysed for p21, p53 and COX2-LI at the IF and TC, and results were correlated with the clinicopathological features. Results: Significantly higher LI was seen in IF compared with TC for p21, p53 and COX2. COX2-LI-IF showed significant positive correlations with perineural and lymphatic invasion, higher T and N stages, and non-papillary gross morphology. Gall-bladder cancers with COX2-LI-IF≥30% showed decreased overall and disease-free survival, and COX2-LI-IF≥30% was an independent poor prognostic factor on multivariable analysis. Gall-bladder cancers with high COX2 and p21-LI-IF showed decreased overall and disease-free survival. Conclusions: The IF of gallbladder cancer is characterised by significantly increased expression of p21, p53 and strong COX2 expression, and strong cytoplasmic COX2 expression at IF is associated with a poorer prognosis. Heterogeneity between TC and IF should be considered in in situ molecular studies, especially during interpretation of immunohistochemical stain results and tissue microarray construction.