Uptake of substrate and electric charge was measured simultaneously in voltage-clamped Xenopus oocytes expressing rat organic cation transporter 2 (rOCT2). At 0 mV, saturating substrate concentrations induced uptake of more positive elementary charges than of monovalent organic cations, with charge-to-substrate ratios of 1.5 for guanidinium⁺, 3.5 for tetraethylammonium⁺, and 4.0 for 1-methyl-4-phenylpyridinium⁺. At negative holding potentials the charge-to-substrate ratios decreased towards unity. At 0 mV, charge-to-substrate ratios higher than unity were observed at different extracellular pH and after replacement of extracellular Na⁺, K⁺, Ca²⁺, Mg²⁺, and/or Cl^-. Charge-to-substrate ratios were not influenced by intracellular succinate^2- or glutarate^2-. The effects of membrane potential and ion substitution strongly suggest that the surplus of transported positive charge is not generated by passive ion permeabilities. Rather we hypothetize that small cations are taken up together with organic cation substrates whereas the outward reorientation of the empty transporter is electroneutral. Nonselective cotransport of small cations was supported by the three-dimensional structures of rOCT2 in its inward-facing and outward-facing conformations which we determined by homology modelling based on the known corresponding structures of H⁺-lactose permease of E. coli, and by functional analysis of OCT mutants. In our model, the innermost cavity of the outward-open binding cleft is negatively charged by Glu448 and Asp475 whereas the inward-open inner cavity is electroneutral containing Asp379, Asp475, Lys215 and Arg440. Substitution of Glu448 by glutamine reduced the charge-to-TEA⁺ ratio at 0 mV to unity. The observed charge excess associated with organic cation uptake into depolarized cells may contribute to tubular damage in renal failure.