A powerful non-adrenergic, non-cholinergic inhibitory innervation of the rabbit rectococcygeus was unmasked by atropine in preparations in which tone had been raised either by carbachol or prostaglandin E2-Flourescent histochemical localization of quinacrine (which binds ATP) revealed ganglia and associated nerve bundles which were neither adrenergic nor cholinergic both on the surface and within the rectococcygeus muscle. Treatment of the rabbit with 6-hydroxydopamine abolished catecholamine fluorescence without affecting quinacrine-stained neural elements. Release of ATP from the rectococcygeus increased 2-4 times above background levels during stimulation of intramural inhibitory nerves. In preparations in which the tone was raised, ATP caused an inhibitory response comparable to that produced by non-adrenergic, non-cholinergic nerve stimulation. Indomethacin reduced the increase in tone and spontaneous activity following washout of ATP in low tone preparations, and potentiated the relaxations produced by ATP in high tone preparations. These results suggest that the rabbit rectococcygeus, as well as receiving a cholinergic excitatory innervation and an adrenergic inhibitory innervation, is also innervated by purinergic inhibitory nerves.