Although nitric oxide (NO) is produced by discrete groups of neurons in the brain, participation of NO in premotor structures directly involved in reflexively evoked, sensory-motor functions has not been demonstrated so far. We now show that NO is a physiological mediator in the generation of a specific motor response in alert behaving animals. In the oculomotor system, numerous neurons expressing nitric oxide synthase (NOS) are located in the prepositus hypoglossi, a nucleus involved in the control of horizontal eye movements. Unilateral inhibition of NOS within this nucleus results in severe ocular nystagmus with slow phases directed to the contralateral side. Accordingly, local increases of NO or cyclic GMP produced a nystagmus in the opposite direction. It is concluded that a balanced production of NO by prepositus hypoglossi neurons is a necessary condition for the normal performance of eye movements in alert animals.