Vestibular compensation following unilateral labyrinthectomy is associated with modifications of the membrane and firing properties of central vestibular neurons. To determine whether gap junctions could be involved in this process, immunofluorescent detection of neuronal connexin 36 and astrocytic connexin 43 was performed in the medial vestibular nucleus (MVN) of rats. In non-lesioned animals, strong staining was observed with anti-connexin 43 antibodies, while moderate staining was obtained with the anti-connexin 36 antibody. However, the expression of either type of connexin was not modified following unilateral labyrinthectomy. These morphological observations were complemented by pharmacological tests performed during extracellular recordings of MVN neurons in guinea pig brainstem slices. In non-lesioned animals, the gap junction blocker carbenoxolone reversibly decreased or suppressed the spontaneous discharge of about 60% of MVN neurons. This reduction was often associated with a long-duration disruption of the regularity of spike discharge. Both effects were mimicked by several other gap junction blockers, but not by glycyrrhizic acid, an analog of carbenoxolone that does not block gap junctions but reproduces its non-specific effects, nor by the selective inhibitor of astrocytic connexin-based networks endothelin-1. Similar effects of carbenoxolone were obtained on the spontaneous activity of ipsilesional MVN neurons recorded in brainstem slices taken from labyrinthectomized animals. Altogether, these results suggest that neuronal gap junctions are involved in shaping the spontaneous activity of MVN neurons. However, unilateral labyrinthectomy does not affect the expression of gap junctions in vestibular nuclei nor their implication in the regulation of neuronal activity.