This study aimed to investigate whether different calcium-binding proteins are present in morphologically and functionally separate cell groups in the dorsal column nuclei of rats. Thalamic-projecting neurons were identified by iontophoretic injection of an intraaxonal tracer substance, choleragenoid, into the ventroposterolateral thalamic nucleus, which was localized by extracellular recordings of the responses to natural peripheral stimulation. The presence of the calcium-binding proteins calbindin and paravalbumin in the projection neurons was detected by a double-labelling immunofluorescent method. The vast majority of the thalamic-projecting neurons contained paravalbumin, but not all parvalbumin-immunoreactive cells were retrogradely labelled. Calbindin-immunoreactive neurons were also found in the dorsal column nuclei, but only a small minority of these neurons projected to the thalamus. These findings are generally consistent with the notion that the different calcium-binding proteins represent functionally separate neuronal populations. Taken together with previous observations that parvalbumin is present in large dorsal root ganglion cells, which project to the dorsal column nuclei, and in the thalamocortical relay cells that receive dorsal column nuclear input, the present findings suggest that parvalbumin is associated with neurons that transmit modality-specific low-threshold mechanoreceptive information from the periphery to the somatosensory cortex. However, the presence of parvalbumin-immunoreactive cells that appeared not to project to the thalamus, as well as the occurrence of thalamic-projecting calbindin-immunoreactive neurons, indicate that parvalbumin and calbindin are present within several, functionally different, groups of neurons in the dorsal column nuclei.