Immunocytochemical staining with antisera directed against substance P (SP) demonstrated the existence of numerous immunoreactive neurons throughout the mediolateral and rostrocaudal extents of the stratum griseum superficiale (SGS) of the superior colliculus (SC) of both rat and hamster. In both of these species, very dense SP-like immunoreactivity (SPLI) was also visible in the parabigeminal nucleus. Combination of retrograde tracing with True blue or Fluorogold and immunocytochemistry demonstrated that SP-positive SC neurons projected to the parabigeminal nucleus in both hamster and rat. Retrogradely labelled and double-labelled cells were most numerous in the rostromedial portion of the SC and rare in the caudal portion of the colliculus. Destruction of the superficial layers of the SC resulted in a virtually complete loss of SPLI in the ipsilateral parabigeminal nucleus in both species. SPLI was also visible in two other targets of the superficial SC laminae: the intergeniculate leaflet and the ventral lateral geniculate nucleus. Ablation of the dorsal SC laminae did not reduce SPLI in either of those nuclei. Our results thus indicate that at least some tectoparabigeminal neurons in hamster and rat contain SPLI and further that the SC appears to be the sole source of SP-positive input to this nucleus.