The putative anion channel mCLCA3 (alias gob-5) is the third murine member of the recently discovered family of calcium-activated chloride channels (CLCA family). Preliminary data suggest that mCLCA3 may play a significant role in diseases with secretory dysfunctions, including asthma and cystic fibrosis. In this study, the mCLCA3 protein was characterized biochemically and its cellular and subcellular distribution pattern was established in normal murine tissues. Polyclonal rabbit antibodies were generated and affinity-immunopurified using synthetic oligopeptides corresponding to the extracellular amino terminus of the mCLCA3 polypeptide. After in vitro translation and glycosylation, proteinase K protection assay, and heterologous expression in COS-7 or HEK 293 cells, SDS-PAGE and immunoblotting revealed a protein structure similar to that of previously characterized CLCA proteins. A systematic light, confocal laser scanning, and transmission electron microscopic immunolocalization study, including virtually all murine tissues, identified the mCLCA3 protein exclusively associated with mucin granule membranes of gastrointestinal, respiratory, and uterine goblet cells and other mucin-producing cells. The results suggest that mCLCA3 may be involved in the synthesis, condensation, or secretion of mucins.