1. The relaxant actions of acetylcholine, substance P and calcitonin gene-related peptide, and the levels of neuropeptide Y and calcitonin gene-related peptide were assessed in the rabbit central ear artery 1, 4 and 6 weeks after a single dose of 45 Gy X-irradiation, a dose similar to that used clinically in intraoperative radiotherapy. 2. Relaxant responses induced by acetylcholine and substance P (both endothelium-dependent) and calcitonin gene-related peptide (endothelium-independent) were reduced, and endogenous neuropeptide Y and calcitonin gene-related peptide levels were unaffected after X-irradiation. 3. The mechanism(s) by which a single dose of 45 Gy X-irradiation may selectively damage relaxant, but not direct, contractile responses of the smooth muscle (as we have shown previously) of the rabbit central ear artery are discussed.