BACKGROUND AND PURPOSE The P2X7 receptor is implicated in inflammation and pain and is therefore a potential target for therapeutic intervention. Here, the development of a native tissue radioligand binding, localization and ex vivo occupancy assay for centrally penetrant P2X7 receptor antagonists is described. EXPERIMENTAL APPROACH Autoradiography studies using the P2X7 antagonist radioligand 3H‐A‐804598 were carried out in rat brain and spinal cord. Subsequent in vitro binding and ex vivo occupancy assays were performed using rat cortex homogenate. KEY RESULTS P2X7 expression was shown to be widespread throughout the rat brain, and in the grey matter of the spinal cord. In binding assays in rat cortex homogenate, ∼60% specific binding was achieved at equilibrium. In kinetic binding assays, kon and koff values of 0.0021·min−1·nM−1 and 0.0070·min−1 were determined, and the Kd derived from kinetic measurements was consistent with that derived from saturation analysis. Novel P2X7 antagonists inhibited the binding of 3H‐A‐804598 to rat cortex P2X7 receptors with Ki values of <40 nM. In an ex vivo occupancy assay, a P2X7 antagonist dosed orally to rats caused a concentration‐dependent inhibition of the specific binding of 3H‐A‐804598 to rat cortex. CONCLUSIONS AND IMPLICATIONS The present study describes the development of an assay that allows localization of P2X7 receptors, the measurement of the binding affinity of P2X7 receptor antagonists in native tissue, and provides a means of determining central P2X7 receptor occupancy. These assays could form an important part of a P2X7 drug discovery programme.