Mucin glycoproteins are a heterogeneous family of high-molecular-mass, heavily glycosylated proteins differentially expressed in epithelial tissue of the gastrointestinal, reproductive and respiratory tracts. We report here the cloning of a mouse caecal mucin (MCM). Amino acid analysis of purified MCM revealed a high content of serine (10.8%) and threonine (25.1%). Antibodies against deglycosylated MCM were prepared for immunohistochemical analysis and for screening a mouse caecal cDNA library. Immunohistochemical analysis showed strong staining of goblet cells and patchy staining of surface columnar cells in the duodenum, small intestine, caecum, colon and rectum. Screening of a mouse caecal cDNA library yielded clones containing tandem repeats of 18 bp with two predominant peptide sequences of TTTADV and TTTVVV. The tandem repeat domain is followed by 1137 bp of non-repetitive sequence and 521 bp of 3′ untranslated sequence prior to the poly(A) tail. Two cysteine-rich regions lie within the 3′ non-repetitive domain. The arrangement of the cysteines within these regions corresponds to epidermal growth factor-like domains. Following the second cysteine-rich region is a stretch of 19 hydrophobic amino acids which may act as a transmembrane domain or allow for interaction with hydrophobic molecules. Northern blot analysis indicates the mRNA is approximately 13.5 kb with greatest expression in the caecum and lesser amounts in the colon and small intestine. No MCM message is found in mouse stomach, trachea, lung, kidney, oesophagus or pancreas. In situ hybridization studies show that MCM message is expressed at the tips of villi in the intestine and in the upper crypts and surface cells of the caecum and colon. Chromosomal analysis assigns this gene to mouse chromosome 5 in a region of conserved linkage with human chromosome 7, the location of the human MUC3 gene. We conclude that we have identified a mouse caecal mucin which represents the mouse homologue of human MUC3. The mouse MUC3 cDNA sequence suggests that it is a novel non-polymerizing mucin which may participate in membrane or intermolecular interactions through its 3′ non-repetitive region.