BACKGROUND/AIMS The mucin-associated trefoil factor (TFF) peptides are integral to cytoprotection. TFF-3 is aberrantly expressed in colorectal and hepatocellular cancer and associated with an invasive phenotype. TFF-3 is also expressed in normal biliary epithelium. However, its role in biliary cancers is unknown. The biological effects of TFFs may result from EGFR, PI3 kinase, COX-2 and STAT-mediated signaling. We investigated the expression of TFF-3, Erk, Akt, EGFR and COX-2 in biliary cancer. METHODOLOGY Twenty-four consecutive cases of cholangiocarcinoma treated from 1996-2002 were studied. Immunohistochemistry was performed using monoclonal antibodies to TFF-3, EGFR, pEGFR, Erk, pErk, Akt, pAkt and COX-2 using validated techniques. Kendall's tau (exact method) and Pearson correlation test were employed to investigate the associations between biomarkers. RESULTS Median age was 67 years. Surveillance, Epidemiology and End Results (SEER) stage was local in 1, regional in 15 and distant in 8. TFF-3 expression was detected in 19 cases. There were no significant associations between TFF-3 expression and sex, stage, grade, survival or SEER score. Erk expression in the tumor showed a borderline, negative correlation with TFF-3 (Pearson's p= -0.3988, p = 0.0588). CONCLUSIONS TFF-3 is commonly expressed in biliary cancers and may have a negative reciprocal relationship with Erk expression.