Crude preparations of brevetoxin (PBTX) produce airway contraction; however, it is not known if this toxin-induced mechanical response is coupled to changes in airway smooth muscle membrane potential. Membrane potentials and contractility of in vitro canine trachealis smooth muscle preparations were simultaneously measured with a microelectrode and microforce transducer before and during exposure to either the crude toxin (0.01-1.2 micrograms/ml), or the purified fractions PBTX-2 or PBTX-3 (0.01-0.07 micrograms/ml). Membrane potentials in cultured airway smooth muscle-reaggregate preparations were similarly studied. Toxins produced concentration-dependent depolarizations and contractions in in vitro preparations. These responses were not obtained in the presence of either the muscarinic blocking agent atropine, the sodium channel blocker tetrodotoxin (TTX), 0 mM extracellular Ca2+, or the Ca2+ channel blocker verapamil. The toxins were without effect in cultured cells, whereas acetylcholine produced depolarizations which were blocked in the presence of atropine, but not TTX. This suggested the presence of functional cholinergic receptors in cultured cells, and the PBTX-induced release of endogenous acetylcholine from peripheral nerve endings in the in vitro airway smooth muscle response.