The regulation of acetylcholine (ACh) lifetime by acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) was evaluated in vitro in canine tracheal smooth muscle preparations. Selective inhibition of AChE by low concentrations of 1,5-bis(N-allyl-N,N-dimethyl-4-ammoniumphenyl)-pentane-3-one dibromide (BW 284C51) led to increases in the amplitude and half-relaxation time of contractions elicited by electric field stimulation. Maximal responses were observed in the presence of 10(-6) M BW 284C51, where the amplitude and half-relaxation time were increased by 84 and 198%, respectively. Higher concentrations of BW 284C51, on the other hand, depressed the amplitude and shortened the decay of electric field stimulation-induced contractions by a mechanism involving blockade of muscarinic receptors. Selective inhibition of BuChE by tetraisopropylpyrophosphoramide (iso-OMPA) led to monotonic increases in the electric field stimulation amplitude and duration. These alterations were less marked than those observed in the presence of BW 284C51. Co-application of BW 284C51 (10(-5) M) and iso-OMPA (10(-5) M) resulted in a 1330% prolongation in the decay of electric field stimulation-induced contractions and the development of a sustained contracture. Such contractures were not observed with either inhibitor alone at any concentration tested. The results indicate that both hydrolytic enzymes are involved in the regulation of ACh lifetime at the canine tracheal neuroeffector junction with AChE exerting the more prominent role. The finding that BuChE co-regulates ACh lifetime in canine trachealis muscle demonstrates a functional role for this enzyme.