The trefoil peptide family encompasses a group of small proteins that appear to assume a distinctive secondary structure that leads to intrinsic resistance to protease digestion. Induction of these peptides has been associated with response to injury in the gastrointestinal tract and related organs. Using an oligonucleotide derived from N-terminal amino acid sequencing of a transformed growth-inhibiting protein, a cDNA was cloned from rat intestinal villus epithelial cells that encodes a protein 81 amino acids in length with the characteristic trefoil peptide cysteine residue motif. Northern blot analysis demonstrates specific expression of a single transcript of 0.43 kilobase in small and large intestinal epithelium in rat and man. Indirect immunofluorescent staining with antiserum raised using a synthetic peptide based on the predicted C-terminal sequence of this protein, designated intestinal trefoil factor, demonstrated that it is primarily expressed and secreted onto the intestinal surface by goblet cells, suggesting that it may be an important component of intrinsic mechanisms for defending mucosal integrity.