We investigated the electrophysiological signature of neuroactive steroid interactions with the plasma membrane. We found that charged, sulfated neuroactive steroids, those that exhibit noncompetitive antagonism of GABA A receptors, altered capacitive charge movement in response to voltage pulses in cells lacking GABA receptors. Uncharged steroids, some of which are potent enhancers of GABAA receptor activity, produced no alteration in membrane capacitance. We hypothesized that the charge movements might result from physical translocation of the charged steroid through the transmembrane voltage, as has been observed previously with several hydrophobic anions. However, the charge movements and relaxation time constants of capacitive currents did not exhibit the Boltzmann-type voltage dependence predicted by a single barrier model. Further, a fluorescently tagged analog of a sulfated neurosteroid altered membrane capacitance similar to the parent compound but produced no voltage-dependent fluorescence change, a result inconsistent with a strong change in the polar environment of the fluorophore during depolarization. These findings suggest that negatively charged sulfated steroids alter the plasma membrane capacitance without physical movement of the molecule through the electric field.