The effect of a synthetic polyanion of Mr 10,000 (a copolymer of methacrylate, maleate and styrene in a 1:2:3 proportion) was studied on isolated rat liver mitochondria and on mitochondrial porin reconstituted into lipid bilayer membranes. Increasing concentrations of the polyanion inhibited the adenyl kinase located between both mitochondrial membranes in a dose-dependent fashion. Upon addition of the detergent digitonin in increasing concentrations the adenyl kinase activity was fully reversible. In reconstitution experiments with mitochondrial porin the polyanion increased the voltage dependence of the pore in such a way that the pore is switched into the closed state at much smaller voltages than in the absence of the polyanion. The asymmetric addition of the polyanion resulted in an asymmetric shift of the voltage-dependence of the pore. If the voltage is negative at the cis-side (the side of the addition of the polyanion) the pore closed rapidly whereas it was always open for potentials of opposite polarity. The results are discussed on the basis of a modification of the gate properties of the mitochondrial porin by the polyanion and by the assumption that the closed state of the pore is not permeable for nucleotides.