The effects of the potassium channel inhibitor and putative botulinum toxin antagonist 3,4-diaminopyridine (3,4-DAP) were investigated in vitro on the contractile properties of rat diaphragm muscle. In the presence of 100 pM botulinum neurotoxin A (BoNT/A), twitches elicited by supramaximal nerve stimulation (0.1 Hz) were reduced to approximately 10% of control in 3 hr at 37 degrees C. Addition of 3,4-DAP led to a rapid reversal of the BoNT/A-induced depression of twitch tension. In the presence of 100 microM 3,4-DAP, antagonism of the BoNT/A-induced blockade began within 30-40 sec and reached 82% of control with a half-time of 6.7 min. The beneficial effect of 3,4-DAP was well maintained and underwent little or no decrement relative to control for at least 8 hr after addition. Application of 1 microM neostigmine 1 hr after 3,4-DAP led to a further potentiation of twitch tension, but this action lasted for < 20 min. Moreover, neostigmine caused tetanic fade during repetitive stimulation. In contrast to the efficacy of the parent compound, the quaternary derivative of 3,4-DAP, 3,4-diamino-1-methyl pyridinium produced little or no twitch potentiation up to a concentration of 1 mM. The potassium channel blocker, tetraethylammonium, generated a transient potentiation followed by a sustained depression of twitch tensions. It is concluded that 3,4-DAP is of benefit in antagonizing the muscle paralysis following exposure to BoNT/A. Co-application of neostigmine or tetraethylammonium with 3,4-DAP, however, appears to confer no additional benefit.