Nitric oxide (NO) containing (nitrergic) interneurons are well-positioned to convey the cortical influences that are crucial for multisensory integration in superior colliculus (SC) output neurons. However, it is not known whether nitrergic interneurons are in this position early in life, and might, therefore, also play a role in the functional maturation of this circuit. In the present study, we investigated the postnatal developmental relationship between these two populations of neurons using Beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH) histochemistry and SMI-32 immunocytochemistry to label presumptive interneurons and output neurons, respectively. SMI-32 immunostained neurons were proved to mature and retained immature anatomical features until approximately 8 postnatal weeks. In contrast, nitrergic interneurons developed more rapidly. They had achieved their adult-like anatomy by 4 postnatal weeks and were in a position to influence the dendritic elaboration of output neurons. It is this dendritic substrate through which much of the cortico-collicular influence is expressed. Double-labeling experiments showed that the dendritic and axonal processes of nitrergic interneurons already apposed the somata and dendrites of SMI-32 labeled neurons even at the earliest age examined. The results suggest that nitrergic interneurons play a role in refining the cortico-collicular projection patterns that are believed to be essential for SC output neurons to engage in multisensory integration and to support normal orientation responses to cross-modal stimuli.