1. The quaternary ammonium compound, N,t-butyl-5-methyl isoxazolium perchlorate (BIP), an anionic group reagent, initially causes contractions of the rat phrenic-nerve diaphragm, guinea-pig ileum and rabbit aortic strip preparations in vitro.2. In addition, the drug produces an irreversible block of indirectly elicited twitch responses in the diaphragm and of contractions induced by acetylcholine, methylfurmethide, dimethyl-phenylpiperazinium, 5-hydroxytryptamine (5-HT), histamine, angiotensin and pilocarpine in the ileum, while direct electrical stimulation of the diaphragm and contractions of the ileum to Ba and K ions are relatively unaffected.3. BIP is also an irreversible inhibitor of acetylcholinesterase but not of butyrylcholinesterase.4. On rabbit aortic strip preparations, responses to histamine, noradrenaline and 5-HT were differentially sensitive to irreversible blockade by BIP.5. Diphenhydramine, used in conditions which gave complete protection of the histamine response to irreversible block by dibenamine, did not protect against the blocking action of BIP but increased the blockade.6. These results suggest that BIP reacts covalently with anionic groups which mediate receptor initiated stimuli. The isoxazolium group may be useful in conferring irreversible properties by its substitution in drug molecules for the pyrrole or pyrrolidine group.