HT-29 cells resistant to 10(-6) M methotrexate (HT29-MTX) secrete mucins with gastric immunoreactivity (Lesuffleur, T., Barbat, A., Dussaulx, E., and Zweibaum, A. (1990) Cancer Res. 50, 6334-6343). A 3310-base pair mucin cDNA clone (L31) was isolated from an HT29-MTX expression library using a polyclonal serum specific for normal gastric mucosa. It shows a high level of identity (98.6%) to clone NP3a isolated from a nasal polyp cDNA library (Meerzaman, D., Charles, P., Daskal, E., Polymeropoulos, M. H., Martin, B. M., and Rose, M. C. (1994) J. Biol. Chem. 269, 12932-12939). However, as a result of changes in reading frame, the 1042-amino acid deduced peptide contains four regions of a low similarity to the NP3a peptide. The amino acid sequence shows 36.3% similarity to part of the carboxyl-terminal sequence of MUC2 including the so-called D4 domain and 21.3% to the pro von Willebrand factor. A short amino acid sequence is similar to cysteine-rich sequences repeated in tracheobronchial, gastric, and colonic mucin cDNAs. The gene corresponding to L31 is located in the mucin gene cluster on chromosome 11p15.5. The patterns of mRNA expression were indistinguishable from those revealed with the JER58 probe (MUC5AC). Southern blot analysis indicates that the L31 and JER 58 sequences are within 20 kilobase pairs of each other. Together, these results suggest that L31 clone is the 3' end of MUC5AC.