The binding of Cu2+ and Mn2+ to the ionophore A23187 in chloroform, 90% ethanol, and sonicated phospholipid dispersions in aqueous mediums has been investigated with electron paramagnetic resonance (epr). The spectra indicated axial symmetry for the Cu2+ complexes and distorted octahedral for the Mn2+ complexes. The coordination between metal ion and its ligands is predominantly ionic in character. The stoichiometry, at the concentrations employed, was found to be 1:2 M2+/ionophore except in 90% ethanol where evidence existed for the 1:1 Cu-A23187 complex, as well. Through competition with Mn2+, the sequence of relative affinities in 90% ethanol was measured to be: Mn2+ greater than La3+ greater than Cu2+ greater than Ca2+ greater than Mg2+ greater than Sr2+. The K A of Mn-A23187 binding is greater than 10 10 M-2. In phospholipid dispersions the spectral characteristics of the Cu complex, particularly g, were observed to be a sensitive function of the hydrocarbon chain mobility. This allowed a calculation of the rotational correlation time of the complex to be made. In sonicated dipalmitoyllecithin was computed to be 10-9 sec, reflecting a local viscosity similar to that sensed by the nitroxide spin-label 2,2,6,6-tetramethylpiperidin-1-oxyl. In a (1:1) lecithin-cholesterol dispersion the complex was significantly more immobilized.