The superior colliculus is the source of a prominent descending pathway which crosses the midline in the mesencephalon and projects to the paramedian pontine reticular formation. The primary goal of the present study was to identify the cells in the superior colliculus of the grey squirrel which give rise to this pathway by using a combination of anterograde and retrograde tracing techniques. Results from the anterograde studies demonstrated the course and terminal distribution of this pathway and suggested that its laminar origin is the intermediate grey layer, stratum griseum intermediale. The retrograde studies were used to confirm the results of the anterograde experiments and to provide a more quantitative estimate of the laminar distribution of the cells which give rise to this pathway. In most cases, over 90% of the cells retrogradely labeled following injections of horseradish peroxidase along the course of this pathway were located in the intermediate grey lamina. This origin is in contrast to that of the ipsilateral tectoreticular pathway which originates primarily in stratum griseum profundum (Holcombe, V., and W. C. Hall (1981) Neuroscience 6: 255–260) and suggests that these two grey layers of the deep tectum are functionally distinct.