AbstractFourier‐transform infrared spectroscopy is applied to examine interactions between divalent metal ions (M2+) and the COO− groups in the M2+‐binding sites of calmodulin as well as effects of M2+ binding on the main chain conformation. Bands due to the symmetric and antisymmetric stretches of the COO− groups in the amino‐acid side chains give information on the coordination of the COO− groups to M2+, and the amide‐I' bands are useful for studying changes in the secondary structure of the protein main chain upon M2+ binding. Infrared deconvolved spectra as well as second‐derivative spectra are sensitive to structural differences among the M2+‐bound forms (M2+ = Mg2+, Ca2+, Sr2+, and Cd2+). Bands at about 1658, 1553, and 1424 cm−1 are characteristic of the Ca2+‐bound form, and are called, respectively, marker bands I, II, and III of the active‐type protein. The Sr2+‐ and Cd2+‐bound forms show deconvolved and second‐derivative spectra similar to those of the Ca2+‐bound form, whereas the Mg2+‐bound form gives second‐derivative and deconvolved spectra close to those of the inactive M2+‐free form. Broad parallelism is found to exist between the marker‐band intensities of the M2+‐bound forms and the abillities of M2+ to stimulate calmodulin‐dependent phosphodiesterase activity. Assignments of the marker bands are discussed. © 1995 John Wiley & Sons, Inc.