Site‐directed mutagenesis was employed to map and characterize Ca²⁺‐binding sites in annexin II, a member of the annexin family of Ca²⁺– and phospholipid‐binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca²⁺ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14732–14739] that a Ca²⁺–binding site is present in the third of the four repeat segments which comprise the 33‐kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca²⁺–binding site in solution is very similar, if not identical, to that of Ca²⁺ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., Römisch, J. and Paques, E.‐P. (1990) FEBS Lett. 275, 15–21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C‐terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high‐affinity Ca²⁺ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca²⁺ sites. Ca²⁺–binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca²⁺ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca²⁺, indicating the presence of (an) additional Ca²⁺ site(s) of presumably different architecture.