The effects of surface potential and transmembrane potential on the binding and fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) in suspensions of rat liver mitochondria was investigated. The binding of ANS is characterized by two classes of binding site: a high affinity (Kd = 10-50 microM), low capacity (n = 3-8 nmol/mg of protein) class in which bound ANS fluoresces strongly, and a low affinity (greater than 500 microM), high capacity (greater than 50 nmol/mg of protein) class with little fluorescence. The dissociation constant, Kd, of the high affinity site strongly depends on the surface potential of the external surface of the inner mitochondrial membrane. Hence, the binding and fluorescence of ANS can be used to estimate the surface potential. The dependence of ANS binding on the medium salt concentration is compatible with the Gouy-Chapman theory and allows accurate determination of surface potential and surface charge. The generation of transmembrane potential, either by oxidizable substrates, ATP, or potassium gradient leads to a decrease in the fluorescence. This decrease is the result of reduced ANS binding. However, the external surface potential as estimated from the charge screening effect of salt solutions is unchanged in energized membranes. The extent of decreased fluorescence correlates reasonably well with the magnitude of the transmembrane potential. The potential-induced quenching depends on pre-equilibration of ANS with the mitochondria, suggesting that the response is due to extrusion of ANS from the mitochondrial matrix. These findings do not support the suggestion that ANS quenching in energized mitochondria is due to an increase in the negative surface charge of the cytosolic surface of the inner membrane. The results are compatible with the suggestion that the response to energization is largely due to the formation of delta psi. However, because of the complex nature of the ANS response, it is concluded that neither the magnitude of surface potential nor the magnitude of membrane potential can be determined from the ANS response in energized mitochondria.