Increase in peripheral chemoreceptor reflex sensitivity and sympathetic nerve activity (SNA) are associated with the development and maintenance of hypertension. Previously, we have shown that denervation of the carotid body (CB) in spontaneously hypertensive (SH) rats reduces arterial pressure (McBryde et al. 2013). Since ATP is a known mediator of signalling in the human CB (Kahlin et al, 2014) and P2X3 receptors underpin afferent hyperreflexia (Ford et al. 2015), we hypothesized that P2X3 receptor upregulation in the CB contributes to increases in arterial pressure. We have validated the expression of P2X3 receptors in the CB of humans and SH rats and initiated functional studies to assess whether a highly selective non-competitive P2X3 receptor antagonist (AF-219) would reduce chemoreflex hypersensitivity, arterial pressure and sympathetic activity in the SH rat. CBs from twelve human cadavers and 12 rats were used for analysis. In human and rat CB, western blotting and immunohistochemistry were performed for the level of expression and localisation of P2X3 receptor protein, respectively. Hypertensive and normotensive rats were implanted with radio-telemetry devices to record arterial pressure (AP) and renal SNA (RSNA) in conscious animals. Animals were infused with AF-219 (8 mg/kg/h) or vehicle i.v. for 1h. The CB was stimulated with sodium cyanide (120μg/kg i.v.). CBs from both human and rats expressed P2X3 receptors. P2X3 receptor protein from the CB was upregulated four fold in SH vs Wistar rat (P<0.05). Glomus cells of human and rat were identified as expressing tyrosine hydroxylase. P2X3 receptor immunofluorescence was detected encapsulating glomus cells. In SH rats, infusion of AF-219 reduced: CB hyperreflexia (P<0.05); AP (systolic -28±3mmHg; P<0.001) and RSNA (-34%; P<0.01). No such changes were detected after vehicle or AF-219 infusion in Wistar rats. In conclusion, P2X3 receptors are present in human CB. In SH rats, P2X3 receptors are upregulated in the CB and contribute to chemoreflex hypersensitivity. Antagonism of P2X3 receptors systemically lowered both AP and RSNA substantially in SH but not Wistar rats. Antagonism of P2X3 receptors in humans as a novel approach for controlling hypertension awaits a clinical trial.