The photoprotein aequorin, capable of emitting light in the presence of a trace amount of Ca2+, is a useful indicator for studying intracellular calcium. The primary structure of aequorin indicated the presence of three Ca(2+)-binding sites, whereas log-log plots of the luminescence intensity versus Ca2+ concentration gave slopes ranging from 2 to 3 depending on the conditions used, suggesting the involvement of two or three Ca2+ ions in the luminescence reaction. Accurate information on the stoichiometry of Ca2+ is essential in interpreting the assay results obtained with aequorin. This study clearly shows that aequorin luminescence is triggered by the binding of two Ca2+ ions, based on the results of titrating aequorin with Ca2+.