Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected unilaterally into the pretectocollicular region of opossums (Didelphis marsupialis aurita), primarily to investigate the existence of a commissural subcortical pathway but also to reveal afferents and efferents of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in this species. Labelled cells and terminals were observed in the contralateral NOT-DTN. Furthermore, HRP was injected bilaterally in the region of the inferior olive (IO) to verify if the distribution of labelled cells in the NOT-DTN overlapped the region of commissural labelled cells. The two subpopulations of retrogradely labelled cells coincided, being distributed within the retinal terminal field attributed to the NOT-DTN, as revealed by contralateral eye injections of HRP. The commissural cells were located slightly more ventral than the olivary cells in the optic tract. The pretectocollicular WGA-HRP injections also labelled cells and terminals bilaterally in the lateral terminal nucleus (LTN), interstitial nucleus of the superior fasciculus, posterior fibers (INSFp), ventral lateral geniculate nucleus (vLGN), and superior colliculus (SC) and ipsilaterally in the medial terminal nucleus (MTN). In addition, further caudally, labelled cells and terminals were observed bilaterally in the nuclei prepositus hypoglossi (PH) and in the medial (MVN) and lateral (LVN) vestibular nuclei. Labelled terminals were found in the ipsilateral nucleus reticularis tegmenti pontis (NRTP) and in the IO with ipsilateral predominance. This study allowed an anatomical delimitation of the NOT-DTN in this opossum species, as defined by the olivary and commissural subpopulations, as well as a hodological evaluation of this region. The existence of some common anatomical aspects with other mammalian species is discussed.