Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.