The actions of bis-ammonium compounds on acetylcholine-activated channels were studied in voltage-clamped neurons of isolated superior cervical ganglion in a rabbit. The kinetics of the compound binding to open channels was estimated from shortening of the decay of fast excitatory postsynaptic current (which is determined by the rate of channels closure). The kinetics of dissociation of the compound from open channels was estimated from the kinetics of the restoration of the second response to double-pulse application of acetylcholine in presence of the blocking compound. The rate constant of bindings of the bis-ammonium compounds to open channels increased while the rate constant of dissociation decreased with membrane hyperpolarization. This voltage-dependence increased with the lengthening of the polymethylene chain in the compound and remained unchanged with the lengthening of nitrogen group radicals. The ganglion-blocking activities of the compounds as determined in the cat ganglion in situ correlated with their rate constants of binding to open channels. It was concluded that ganglion-blocking actions of bis-ammonium compounds is determined by their channel-blocking activities.