Three morphologically distinct types of lamina I neurones, fusiform, flattened and pyramidal, project from the spinal cord to the caudal part of the nucleus tractus solitarii in the rat, and may represent a pathway whereby peripheral stimuli can modify autonomic functions. The neurochemistry of these three types of projection neurones was investigated using retrograde neuronal tracing with cholera toxin B-subunit combined with dual and triple immunofluorescence labelling for different neuroactive substances. None of the lamina I neurones with immunoreactivity for GABA or glycine were found to project to the nucleus tractus solitarii, whereas high levels of glutamate immunoreactivity, which may indicate a glutamatergic phenotype, were found in 18.4% of fusiform, 9.6% of pyramidal and 2.1% of flattened projection neurones. Immunoreactivity for calbindin-D28K was present in 34.9% of fusiform cells, 18.3% of pyramidal cells and 10.5% of flattened cells, and nitric oxide synthase immunoreactivity was detected in 13.8% of fusiform cells, 1.1% of pyramidal cells and 4.2% of flattened cells that had projections to the nucleus tractus solitarii. Calbindin immunoreactivity was co-localised in major subpopulations of projection neurones of each morphological type that contained glutamate immunoreactivity, whereas co-localisation of nitric oxide synthase immunoreactivity in these neurones was relatively uncommon. The pyramidal cell was the only retrogradely labelled cell type found to be immunoreactive for substance P, but few (<5%) of these neurones were immunolabelled. These data are consistent with the hypothesis that lamina I neurones projecting to the dorsal vagal complex are not inhibitory, and that some of them, belonging mostly to the fusiform and pyramidal types, may exert excitatory, glutamate- or substance P-mediated effects upon inhibitory interneurones in the nucleus tractus solitarii. Such excitatory pathways could be involved in the attenuation of the reflex control of blood pressure by both painful and innocuous peripheral stimuli, such as those arising in injury and exercise.