The effects of drugs and electrical field stimulation on muscle strips from the human internal anal sphincter have been examined to provide information about the receptors and nerves that might be involved in the relaxation of the muscle in vivo. Acetylcholine and bethanechol usually relaxed muscle strips; this effect was abolished by hyoscine and antagonized to a varying degree by tetrodotoxin. Hexamethonium in concentrations sufficient to block relaxations to 1,1-dimethyl-4-phenylpiperazinium iodide or nicotine had no effect on relaxations due to acetylcholine, thereby indicating that acetylcholine was acting on muscarinic receptors. The nerves stimulated by acetylcholine released an unknown transmitter. Both 1,1-dimethyl-4-phenylpiperazinium iodide and nicotine relaxed muscle strips, possibly by releasing an adrenergic neurotransmitter which, because the responses to nicotinic receptor-stimulation were blocked by propranolol, stimulated beta-adrenergic inhibitory receptors. Sphincter muscle was also relaxed by electrical field stimulation of intrinsic nerves; this response was blocked by tetrodotoxin but unaffected by hexamethonium, hyposcine, or propranolol. The nerves responding to electrical field stimulation were therefore post-ganglionic, noncholinergic, and nonadrenergic. Compounds discounted as possible neurotransmitters of the noncholinergic, nonadrenergic inhibitory nerves were prostaglandin E2 and F2 alpha, histamine, 5-hydroxytryptamine, and dopamine. Some evidence allows vasoactive intestinal peptide and adenosine triphosphate to be considered as possible neurotransmitters; this could not be confirmed because selective antagonists are not yet available.