Although recent studies implicated glutamate receptors and nitric oxide in nociception, much still needs to be known about their localisation in neurons involved in nociceptive transmission from the orofacial region. In this study, c-fos expression indicated by Fos immunohistochemistry in the caudal spinal trigeminal nucleus induced by subcutaneous injection of formalin into the lateral face of the rat was used as a marker for nociceptive neurons. The study sought to determine whether Fos-positive neurons express nitric oxide synthase, glutamate N-methyl-D-aspartate type receptor subunit 1, and glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type receptor subunit 2/3; and whether they project to the thalamus. After formalin injection, many Fos-positive nuclei appeared in the superficial laminae of the ipsilateral trigeminal nucleus. Confocal laser scanning microscope revealed that almost all neurons with Fos immunofluorescent nuclei were colocalised with N-methyl-D-aspartate receptor 1, 94% with glutamate receptor 2/3 and 14% with nitric oxide synthase. Some of them were closely related to neurons labelled by nitric oxide synthase. Lastly, some of the Fos-positive neurons were labelled by tetramethylrhodamine-dextran injected into the trigeminothalamic tract or the thalamic region. The results suggested that activation of N-methyl-D-aspartate receptor 1 and glutamate receptor 2/3 upon glutamate release in response to noxious stimulation to the orofacial region might mediate c-fos expression in neurons involved in nociception. The expression of Fos in the neurons could also be mediated by nitric oxide produced from the same, as well as neighbouring neurons, when nociceptive stimulation persisted. Fos-positive neurons in the spinal trigeminal nucleus may project to the thalamus, relaying orofacial nociception to the higher sensory centre.