The nucleus of the optic tract (NOT) has been implicated in the initiation of the optokinetic reflex (OKR) and in the modulation of visual activity during saccades. The present experiments demonstrate that these two functions are served by separate cell populations that can be distinguished by differences in both their cellular physiology and their efferent projections. We compared the response properties of NOT cells in rats using target directed whole-cell patch clamp recording in vitro. To identify the cells at the time of the recording experiments, they were pre-labeled by retrograde axonal transport of WGA-apo-HRP-gold (15 nm), which was injected into their primary projection targets, either the ipsilateral superior colliculus (iSC), or the contralateral NOT (cNOT) or the ipsilateral inferior olive (iIO). Retrograde labeling following injections in single animals of either WGA-apo-HRP-gold with different particle sizes (10 nm and 20 nm) or two different fluorescent dyes distinguished two NOT cell populations. One projects to both the iSC and cNOT. These cells are spontaneously active in vitro and respond to intracellular depolarizations with temporally regular tonic firing. The other population projects to the iIO and consists of cells that show no spontaneous activity, respond phasically to intracellular depolarization, and show irregular firing patterns. We propose that the spontaneously active pathway to iSC and cNOT is involved in modulating the level of visual activity during saccades and that the phasically active pathway to iIO provides a short latency relay from the retina to premotor mechanisms involved in reducing retinal slip.