Nitric oxide (NO) production by neurons in the prepositus hypoglossi (PH) nucleus is necessary for the normal performance of eye movements in alert animals. In this study, the mechanism(s) of action of NO in the oculomotor system has been investigated. Spontaneous and vestibularly induced eye movements were recorded in alert cats before and after microinjections in the PH nucleus of drugs affecting the NO-cGMP pathway. The cellular sources and targets of NO were also studied by immunohistochemical detection of neuronal NO synthase (NOS) and NO-sensitive guanylyl cyclase, respectively. Injections of NOS inhibitors produced alterations of eye velocity, but not of eye position, for both spontaneous and vestibularly induced eye movements, suggesting that NO produced by PH neurons is involved in the processing of velocity signals but not in the eye position generation. The effect of neuronal NO is probably exerted on a rich cGMP-producing neuropil dorsal to the nitrergic somas in the PH nucleus. On the other hand, local injections of NO donors or 8-Br-cGMP produced alterations of eye velocity during both spontaneous eye movements and vestibulo-ocular reflex (VOR), as well as changes in eye position generation exclusively during spontaneous eye movements. The target of this additional effect of exogenous NO is probably a well defined group of NO-sensitive cGMP-producing neurons located between the PH and the medial vestibular nuclei. These cells could be involved in the generation of eye position signals during spontaneous eye movements but not during the VOR.