Acetylcholinesterase inhibitors produce diverse physiologic effects, but lethal exposure consistently produces respiratory failure due to neuromascular paralysis or depression of respiratory control centers in the medulla. Simultaneous measurement of gastrocnemius muscle contraction and efferent phrenic nerve activity was used to determine the primary cause of respiratory failure produced by neosligmine and diisopropyi fluorophosphate (DFP) in anesthetized cats. Both neostigmine and DFP abolished phrenic nerve activity prior to producing neuromuscular blockade. Furthermore, neostigmine did not alter brain acetylcholinesterase activity and pretreatment with either atropine methylbromide or atropine increased the dose of neostigmine required to abolish phrenic nerve activity. In contrast, DFP abolished brain cholineslerase activity and only atropine inhibited its respiratory effects. Despite the loss of efferent phrenic nerve activity, there is no evidence of a direct effect of neostigmine on respiratory control centers. Neostigmine may instead alter afferent inputs which modulate respiration to produce a reflex respiratory failure.