Calbindin-D28k is a highly abundant protein found in neurons in selected brain regions, including cells in sensory systems of the brainstem. Because of its capacity to bind cytosolic Ca++, calbindin-D28k is thought to contribute to the regulation of compartmental Ca++ concentrations in neurons. In this study of the rat spinal trigeminal nucleus, calbindin-D28k was localized with immunoperoxidase and immunogold methods. Results showed that immunoreactive calbindin-D28k neurons were widely distributed to all regions of the nucleus, but were particularly numerous in the substantia gelatinosa. Some trigemino-thalamic neurons that were identified by retrograde labeling of a conjugated wheat-germ agglutinin with horseradish peroxidase also contained calbindin-D28k immunoreactivity. Most of the calbindin-D28k labeling was found in cell bodies and dendrites. Axon terminals were rarely stained. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also heavily associated with euchromatin within nuclei. These findings show that immunoreactive calbindin-D28k is localized to both interneurons and projecting neurons of the spinal trigeminal nucleus. Many of these cells are likely to receive glutamatergic afferent inputs, which may act in part by increasing Ca++ flux into the neurons. Calbindin-D28k has a high capacity for buffering Ca++ and under some conditions may protect neurons against glutamate-induced excitotoxicity. We speculate that calbindin-D28k may function to regulate calcium concentrations in spinal trigeminal neurons.