Human intestinal trefoil factor (ITF), a mucosa-associated trefoil peptide, has been implicated in maintenance of mucosal integrity and may denote commitment to goblet cell differentiation. We have quantitated and localized ITF expression in normal and neoplastic colons, determined the molecular forms present, and examined the relationships among ITF expression and mucin production and increasing dysplasia. Normal and neoplastic human colonic mucosa (n = 30) were extracted for quantitation by ITF radioimmunoassay and size determination by gel filtration and immunoblotting. Paraffin sections of normal bowel, hyperplastic polyps, adenomatous polyps, and adenocarcinoma were examined for ITF immunohistochemistry and mucin histochemistry. The predominant molecular species in both normal and neoplastic colon was a 7-kd monomer. Staining was localized to goblet and Paneth cells and the luminal surface in normal colon and in most adenomatous polyps but not hyperplastic polyps; this colocalized with periodic acid-Schiff histochemistry. ITF staining of undifferentiated cells was seen with increasing dysplasia, and Golgi region immunoreactivity was highly conserved in adenocarcinoma, independent of the presence of periodic acid-Schiff-positive mucin. ITF concentration in colonic extracts was 10 to 200 pmol/g. Levels in normal (88.1 +/- 15.1 pmol/g) and malignant (90.1 +/- 12.7 pmol/g) tissue were comparable. In carcinomas, there were significant associations among ITF expression and degree of differentiation and mucin presence. Loss of expression was associated with tumor necrosis and advanced Duke's stage. ITF is uniformly processed in normal and neoplastic colons. Goblet cell-derived ITF is associated with stainable mucin production. ITF synthesis by non-goblet colonocytes, however, is highly conserved in neoplastic differentiation.