Knowledge of the polysynaptic pathway conveying photic information to the pineal gland is based upon studies employing lesions, knife cuts and classical tracers. In the present investigation we used viral transneuronal tracing to re-examine the organization of this circuitry. This was accomplished by injecting a neurotropic alpha herpesvirus (pseudorabies virus) into the gland and localizing viral antigen in infected neurones at various postinoculation intervals. This approach is based upon the demonstrated ability of this virus to invade axon terminals, replicate in neurones and pass retrogradely through a multisynaptic circuit. Immunohistochemical localization of viral antigen revealed the progressive appearance of infected neurones in the superior cervical ganglion (SCG), intermediolateral nucleus of the upper thoracic spinal cord (IML), parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN). Other infected cell groups known to project to the IML also became infected. Infection of the PVN reproducibly involved neurones in the dorsal, medial and lateral parvicellular subdivisions and preceded the appearance of infected neurones in the SCN and other regions of hypothalamus. Topographic analysis of virus infected neurones within the SCN revealed differential infection of SCN subdivisions that suggested topography in the projection of the SCN to the PVN. Removal of the SCG eliminated infection within the aforementioned circuitry and revealed a parasympathetic innervation from the sphenopalatine ganglion. The data provide further detail on the cellular identity and synaptology of neural circuitry controlling the rhythmic secretion of melatonin by the rat pineal gland.