Pilocarpine contracts the sphincter pupillae muscle via an effect on muscarinic receptors and phenylephrine contracts the dilator pupillae muscle via an effect on alpha-adrenergic receptors. These effects are thought to mimic the action of the parasympathetic and sympathetic nervous systems, respectively. Intracellular injection of substance P (SP) produces an atropine-resistant constriction of the pupil. This response is thought to mimic the effect of local sensory reflexes on the sphincter pupillae muscle, involving SP-containing trigeminal nerve endings. Repeated intraocular injections of tetrodotoxin, a general blocker of nervous conduction, over a period of 3 weeks produced supersensitivity to pilocarpine, phenylephrine and SP in the rabbit iris. These findings support the view that, like acetylcholine and noradrenaline, SP or an SP-like compound acts as a neurotransmitter in the iris. Also, long-term topical application of an SP antagonist, (D-Pro2,D-Trp7,9)-SP or (Arg5,D-Trp7,9)-SP5-11, to the rabbit eye produced supersensitivity to SP but not to pilocarpine, thus supporting the view that the SP antagonists interact specifically with the SP receptors. The isolated rabbit iris sphincter muscle responds to electrical stimulation with a cholinergic twitch followed by a slow non-cholinergic contraction that can be blocked by antagonists to SP. Analysis of the motor activity of the iris sphincter muscle after long-term topical treatment of the eye with an SP antagonist followed by an interval of 2 days after termination of treatment revealed a greatly enhanced non-cholinergic contraction compared with the cholinergic twitch, a finding that seems to be consistent with the idea that supersensitivity to SP had developed.