Acetylcholine (Ach) has received strong support as the neurotransmitter at vestibular efferent nerve endings. Ach, cholinomimetics and cholinergic antagonists were therefore applied to frog isolated whole labyrinths and isolated semicircular canals. Both spontaneous and evoked single unit and multiple unit activities were recorded from the decentralized posterior semicircular canal afferent nerve. In a manner analogous to efferent nerve stimulation, Ach produced both facilitatory and inhibitory changes in afferent firing rates. The facilitatory effect is likely mediated by muscarinic receptors (i.e. atropine antagonizes it at low concentrations). The facilitatory effect can also be elicited by muscarine and carbachol and it is likely produced presynaptically on the vestibular sensory cell. That is, the effects of Ach are not changed by removal of the efferent neurons but they are absent when afferent transmitter release is blocked. The inhibitory effect is not as well characterized as is the facilitatory effect but it can be blocked by strychnine. The results are consistent with the hypothesis that Ach is the transmitter responsible for both the facilitatory and the inhibitory effects of efferent vestibular nerve stimulation.