Pulsed-dye laser excitation and lifetime spectroscopy of the 7F0----5D0 transition of Eu3+ reveals details of the binding of this ion to the calcium-binding sites of calmodulin (labeled I-IV, starting at the N-terminus). For 10 microM calmodulin Eu3+ binds quantitatively at sites I and II and more weakly at sites III and IV with Kd values of approximately 0.5 microM and 1.0 microM at the latter sites. In D2O solution the time course of luminescence emission of Eu3+-loaded calmodulin can be separated into three exponential components with lifetimes of 2.50 (sites I and II) and 1.70 and 0.63 ms (sites III and IV). This finding permits the time resolution of the excitation spectrum by determination of the amplitudes of the three components as the excitation wavelength is scanned across the spectral profile in 0.1-nm increments. The amplitudes (intensities at time t = O) are plotted as a function of wavelength and the results fitted to three Lorentzian peaks centered at 579.20, 579.40, and 579.32 nm in order of decreasing lifetimes. In H2O solution only two exponential luminescence decay components are resolvable with lifetimes of 0.41 and 0.27 ms, corresponding to sites I and II and sites III and IV, respectively. These results indicate that two water molecules are coordinated to the Eu3+ ions at sites I and II and at either site III or site IV, with three water molecules at the remaining site.(ABSTRACT TRUNCATED AT 250 WORDS)