Long alkyl chain quaternary ammonium ions (QA), the local anesthetics (LA) tetracaine and lidocaine, imipramine, and pancuronium cause inactivation of the alamethicin-induced conductance in lipid bilayer membranes. The alamethicin-induced conductance undergoes inactivation only when these amphipathic compounds are added to the side containing alamethicin. The concentration of QA required to cause a given amount of inactivation depends on the length of the hydrocarbon chain and follows the sequence C9 greater than C10 greater than C12 greater than C16. LA and imipramine, in contrast to QA or pancuronium, are able to promote appreciable inactivation only if the pH of the alamethicin-free side is equal to or lower than the pK of these compounds. The membrane permeability to QA, LA, or imipramine is directly proportional to the alamethicin-induced conductance and is larger than the one for potassium. The observed steady state and time-course of the inactivation are well described by a model similar to that proposed by Heyer et al. (1976. J. Gen. Physiol. 67:703--729) and extended for any value of the diffuse double layer potential and for LA and imipramine. In this model QA, LA, or imipramine are able to permeate through the membrane only when the alamethicin-induced conductance is turned on. The amphipathic compounds then bind to the other membrane surface, changing the transmembrane potential and turning the conductance off. For a given concentration of QA, LA, or imipramine the extent of inactivation depends on two factors: first, the binding characteristics of these compounds to the membrane surface and second, their ability to permeate through the membrane when the alamethicin-induced conductance is turned on. The several possible mechanisms of permeation of the amphipathic molecules tested are discussed.