Calcium-dependent regulation of intracellular processes is mediated by proteins that on binding Ca2+ assume a new conformation, which enables them to bind to their specific target proteins and to modulate their function. Calmodulin (CaM) and troponin C, the two best characterized Ca2+-regulatory proteins, are members of the family of Ca2+-binding proteins utilizing the helix-loop-helix structural motif (EF-hand). Herzberg, Moult, and James (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) proposed that the Ca2+-induced conformational transition in troponin C involves opening of the interface between the alpha-helical segments in the N-terminal domain of this protein. Here we have tested the hypothesis that a similar transition is the key Ca2+-induced regulatory event in calmodulin. Using site-directed mutagenesis we have substituted cysteine residues for Gln41 and Lys75 (CaM41/75) or Ile85 and Leu112 (CaM85/112) in the N-terminal and C-terminal domains, respectively, of human liver calmodulin. Based on molecular modeling, cysteines at these positions were expected to form intramolecular disulfide bonds in the Ca2+-free conformation of the protein, thus blocking the putative Ca2+-induced transition. We found that intramolecular disulfide bonds are readily formed in both mutants causing a decrease in affinity for Ca2+ and the loss of ability to activate target enzymes, phosphodiesterase and calcineurin. The regulatory activity is fully recovered in CaM41/75 and partially recovered in CaM85/112 upon reduction of the disulfide bonds with dithiothreitol and blocking the Cys residues by carboxyamidomethylation or cyanylation. These results indicate that the Ca2+-induced opening of the interfaces between helical segments in both domains of CaM is critical for its regulatory properties consistent with the Herzberg-Moult-James model.