The superior colliculus (SC) contains two major subdivisions, the superficial layers (sSC) and the deeper layers (dSC). sSC receives visual information from the retina and visual cortex, while dSC sends descending projections to the brainstem and spinal cord. It has not been clear whether and how sSC directly activates dSC, however recent studies in slice preparations reported that electrical stimulation of sSC induces burst firing in dSC neurons after application of bicuculline. In the present study, we tested whether sSC directly activates dSC in vivo. In isoflurane-anesthetized rats, electrical stimulation of the optic nerve (ON) induced negative field responses mainly in sSC, but not as much in dSC, under control conditions. However, after injection of bicuculline into dSC, ON stimulation induced long-lasting negative field responses in dSC. dSC neurons including the tectofugal neurons exhibited burst firing during the long-lasting negative field responses. The burst responses remained after ablation of the cerebral cortex ipsilateral to the SC recordings. These results suggest that retinal inputs induce burst responses in tectofugal neurons in dSC via subcortical pathways when the SC circuit is released from GABA(A) receptor-mediated inhibition. We propose that sSC is the main candidate for the source of the excitatory synaptic inputs to dSC among the subcortical regions.