The multifunctional tissue transglutaminase 2 (TG2) has a four-domain structure with several Ca(2+)-regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure of the Ca(2+)-binding form of the human enzyme is not known, and its Ca(2+)-binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca(2+)-binding residues of epidermal transglutaminase and factor XIIIa (S1-S3), and two regions with negative surface potentials (S4 and S5). CD spectroscopy, antibody-binding assay and GTPase activity measurements indicated that the amino acid substitutions did not cause major structural alterations. Calcium-45 equilibrium dialysis and isothermal calorimetric titration showed that both wild-type and active site-deleted enzymes (C277S) bind six Ca(2+). Each of the S1-S5 mutants binds fewer than six Ca(2+), S1 is a strong Ca(2+)-binding site, and mutation of one site resulted in the loss of more than one bound Ca(2+), suggesting cooperativity among sites. All mutants were deficient in transglutaminase activity, and GTP inhibited remnant activities. Like those of the wild-type enzyme, the GTPase activities of the mutants were inhibited by Ca(2+), except in the case of the S4 and S5 mutants, which exhibited increased activity. TG2 is the major autoantigen in celiac disease, and testing the reactivity of mutants with autoantibodies from celiac disease patients revealed that S4 strongly determines antigenicity. It can be concluded that five of the Ca(2+)-binding sites of TG2 influence its transglutaminase activity, two sites are involved in the regulation of GTPase activity, and one determines antigenicity for autoantibodies in celiac patients.