Intracellular Ca2+ plays an important role in signal transduction, and we are developing new MRI techniques to study its regulation in living animals. We have reported on an MR1 contrast agent (DOPTA-Gd) where the relaxivity of the complex is controlled by the presence or absence of the divalent ion Ca2+. By structurally modulating innersphere access of water to a chelated Gd3+ ion, we observe a substantial and reversible change in T, upon the addition of Ca2+ and not other divalent ions. Luminescence lifetime and NMRD measurements of the complex have been acquired, and several parameters contribute to the Ca2+ dependent relaxivity change of DOPTA-Gd. The number of inner-sphere water molecules is more than doubled after the Ca2+ concentration is increased. This finding strongly supports the proposed conformational change of DOPTA-Gd when Ca2+ is bound. Relaxometric measurements confirm these results and provide an indication that second-sphere water molecules are probably responsible for paramagnetic relaxation enhancement in the absence of Ca2+. After Ca2+ is bound to DOPTA-Gd, the molecule undergoes a substantial conformational change that opens up the hydrophilic face of the tetraazacyclododecane macrocycle. This change dramatically increases the accessibility of chelated Gd3+ ion to bulk solvent. The design of this class of calcium-activated MR contrast agent was based primarily on the assumption that the number of coordinated inner-sphere water molecules would be the dominating factor in observed relaxivity measurements. This result has been confirmed; however, careful mechanistic studies reveal that additional factors are involved in this process.