Cardiopulmonary receptors relay signals to the central nervous system via vagal and spinal visceral afferents. To date there are no detailed topographical studies in mice indicating the distribution of central neurones activated following stimulation of cardiopulmonary afferents. In anaesthetised mice, we injected the 5-HT(3) receptor agonist phenylbiguanide (PBG), a drug that is known to stimulate cardiopulmonary afferent C-fibres, into the right atrium of the heart and mapped c-Fos expression within specific regions of the central nervous system. Intra-atrial injection of PBG produced a reflex cardiorespiratory response including a pronounced bradycardia and a respiratory depression. Using immunohistochemical detection of the protein product of the immediate-early gene c-fos, we mapped the brain regions affected by cardiopulmonary 5-HT(3) receptor stimulation. Within the nucleus of the solitary tract (nTS) of PBG-injected mice, we detected an increased number of c-Fos-positive nuclei in the dorsolateral and gelatinous parts at the level of the area postrema (-7.48 mm bregma) but not at more rostral or caudal levels (-7.76, -7.20, -6.84 and -6.36 mm bregma) relative to vehicle-injected control mice. In addition, c-Fos expression in the crescent part of the lateral parabrachial nucleus was decreased in PBG-injected mice whereas no significant differences were detected between PBG-injected and control mice in the number of c-Fos-positive nuclei in the dorsal part of the lateral parabrachial nucleus. PBG injections had no significant effects on the number of c-Fos-positive catecholaminergic neurones within the C1/A1, C2/A2, A5, A6 and A7 cell groups. Likewise, PBG injections had no significant effects on c-Fos expression in other central regions involved in cardiorespiratory control or cardiorespiratory reflexes (selected non-catecholaminergic nuclei in the medulla and midbrain periaqueductal gray, the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala). Identification of specific regions of the nTS complex involved in relaying signals from afferent cardiopulmonary C-fibres to the central nervous system will be useful for future studies aimed at understanding neural mechanisms underlying cardiopulmonary reflexes and physiological responses to cardiopulmonary disease.