Immunocytochemistry for calbindin (CA) and parvalbumin (PA) was combined with retrograde tracing from the thalamus, superior colliculus (SC), and cerebellum to define the ascending projections of neurons in the rat's trigeminal (V) brainstem complex that express immunoreactivity for these calcium binding proteins. Many PA‐immunoreactive neurons were observed in trigeminal nucleus principalis (PrV). Many of these cells projected to thalamus and a few sent axons to SC. In ventral PrV, PA‐immunoreactive neurons were arranged in a vibrissa‐related pattern. A very small number of large CA‐immunoreactive neurons were observed in dorsomedial PrV. None of these cells were labeled by our tracer deposits. Small neurons in V subnucleus oralis (SpO) were also immunoreactive for PA, but none were retrogradely labeled. A small percentage of the large neurons in SpO were CA‐immunoreactive; many of these were retrogradely labeled by tracer injections in the thalamus and/or SC. In V subnucleus interpolaris (SpI), many small to medium sized cells were PA‐positive and they were arrayed in a vibrissae‐like pattern. None of these neurons were retrogradely labeled from any of the above‐listed targets, but many were retrogradely labeled by tracer injections into ipsilateral PrV. SpI also contained many large CA‐immunoreactive cells. Many of these projected to the thalamus and/or SC and some were also retrogradely labeled by tracer injections into ipsilateral PrV. In V subnucleus caudalis (SpC), very dark PA‐immunoreactive neurons were located in the inner part of lamina II and less often in laminae I. Lightly labeled cells were located in the magnocellular laminae and formed vibrissa‐related aggregates. None of these neurons were retrogradely labeled by our tracer injections. CA‐immunoreactive cells were located throughout the depth of lamina II in SpC and smaller numbers were also visible in lamina I and layers III‐V. A small percentage of the CA‐positive cells in lamina I and in the magnocellular layers were retrogradely labeled from the thalamus. These data indicate that PA and CA antisera identify two cell populations in whisker‐related regions of the V brainstem complex and that PA cells are somatotopically patterned in PrV, SpI, and SpC. These markers also distinguish two cell groups in superficial laminae of the medullary dorsal horn.