Secreted mucins protect the underlying epithelium by serving as the major determinant of the rheological property of mucus secretion and the receptors for pathogens. These functions can be affected by the three branch structures, including core 2, core 4, and blood group I, which are synthesized by the mucus-type core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT-M). Decreased activity of this enzyme and expression of this gene have been found in colorectal cancer, which supports the important role of this enzyme in the protective functions of secreted mucins. We cloned full-length mouse (m) C2GnT-M cDNAs and showed that the deduced amino acid sequence was homologous to those of other C2GnT-Ms. The recombinant protein generated by mC2GnT-M cDNA exhibited core 2, core 4, and blood group I enzyme activities with a ratio of 1.00:0.46:1.05. We identified two different size transcripts by rapid amplification of cDNA ends and RT-PCR. Derived from the 6.6 kb mC2GnT-M gene composed of three exons and two introns, these two transcripts were intronless and differed by the length of the 3' untranslated region. In addition, exon 2 was found to be heterogeneous in size. This gene was highly expressed in the gastrointestinal tract, including colon, stomach, and small intestine. Antibodies generated against mC2GnT-M identified this enzyme in the goblet cells and other mucus cells/glands. This report provides the basis for further characterization of the regulation of mC2GnT-M gene expression and the biological functions of this gene.