The regulatory domain of scallop myosin is a three-chain protein complex that switches on this motor in response to Ca2+ binding. This domain has been crystallized and the structure solved to 2.8 A resolution. Side-chain interactions link the two light chains in tandem to adjacent segments of the heavy chain bearing the IQ-sequence motif. The Ca(2+)-binding site is a novel EF-hand motif on the essential light chain and is stabilized by linkages involving the heavy chain and both light chains, accounting for the requirement of all three chains for Ca2+ binding and regulation in the intact myosin molecule.