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Neural structures have been shown to be present in valve cusp tissue. We aimed to characterise the influence of neuronal stimulation on the component structures of the aortic root and cusps. Specimens of sinus, sinotubular junction (STJ), annulus and cusp tissue were dissected from porcine aortic roots and either stimulated with electrical field stimulation (EFS) in isolated tissue baths or fixed for immunohistochemical characterisation of neuronal structures. Sinus, STJ and annular tissue all gave tetrodotoxin-sensitive, frequency-dependent contractions in response to EFS. Contractions in annular tissue were only evident in tissue from the left- and non-coronary cusps, but not from the right-coronary cusp. Cusp tissue gave no contractile response to EFS, however in the presence of 1 mumol tetrodotoxin a strong contractile response was evident. This contractile response was unmasked when cusp tissue was stimulated in the presence of a nitric oxide synthase or guanylate cyclase inhibitors. Immunohistochemical analysis identified a network of neurofilament positive fibers in tissue from all aortic root structures that were associated with the presence of tyrosine hydroxylase and choline acetyl transferase. The nerve fibers in cusp tissue were in close proximity to the endothelial surface and demonstrated positive staining for neuronal nitric oxide synthase. Nerves in the aortic valve exert a nitric oxide-mediated neurogenic dilator tone in cusp tissue and are capable of producing contractile responses in different components of the aortic root. These responses could influence valve function in health and disease.