Three ionic detergents commonly used in membrane-bound protein isolation and reconstitution experiments, SDS, cholate, and DOC, are shown to act as divalent cation ionophores when incorporated into black lipid membranes made from either oxidized cholesterol or a mixture of phosphatidylcholine and cholesterol (PC/cholesterol = 5:1 mg). At a concentration greater than or equal to 1 microM, SDS shows large selectivity differences between cations and anions and among the different cations tested (Ba2+, Ca2+, Sr2+, Mg2+, and Mn2+). Deoxycholate and cholate at concentrations greater than 4 X 10(-4) M and 10(-3) M, respectively, also act as divalent cation ionophores. The selectivity sequence measured for these two detergents is evidence for a strong ionic interaction between the divalent cation and the anionic charged groups on the detergent. In the case of cholate, the conductance depends on the third or fourth power of the cholate concentration and shows a linear dependence on CaCl2 concentration. The conductance for deoxycholate depends onthe sixth or seventh power of the DOC concentration and is also linearly dependent on the CaCl2 concentration. In an oxidized cholesterol black lipid membrane in the presence of 5 mM CaCl2, small concentrations of LaCl3 (less then 1 microM) inhibit the ionophoric activity of each of the detergents tested. Evidence is presented to show that this inhibitory effect is a nonspecific effect on oxidized cholesterol BLM's, and is not due to a direct effect of La3+ on detergent-mediated transport.