Proteins of the CLCA gene family have been proposed to mediate calcium-activated chloride currents. In this study, we used detailed bioinformatics analysis and found that no transmembrane domains are predicted in hCLCA1 or mCLCA3 (Gob-5). Further analysis suggested that they are globular proteins containing domains that are likely to be involved in protein-protein interactions. In support of the bioinformatics analysis, biochemical studies showed that hCLCA1 and mCLCA3, when expressed in HEK293 cells, could be removed from the cell surface and could be detected in the extracellular medium, even after short incubation times. The accumulation in the medium was shown to be brefeldin A-sensitive, demonstrating that hCLCA1 is constitutively secreted. The N-terminal cleavage products of hCLCA1 and mCLCA3 could be detected in bronchoalveolar lavage fluid taken from asthmatic subjects and ovalbumin-challenged mice, demonstrating release from cells in a physiological setting. We conclude that hCLCA1 and mCLCA3 are non-integral membrane proteins and therefore cannot be chloride channels in their own right.