Acute administration of capsaicin in vitro produced either vascular smooth muscle contraction (cat middle cerebral artery) or smooth muscle relaxation (guinea pig carotid artery and thoracic aorta). Prior in vivo treatment with capsaicin abolished the relaxation response of guinea pig vessels to acute capsaicin. Instead a contractile response was seen after chronic capsaicin treatment, suggesting that the relaxation response produced by capsaicin is due to release of a vasodilator substance. Substance P caused relaxation in both cat cerebral arteries and the guinea pig thoracic aorta, an effect which was abolished or reduced by endothelial damage. However, responses to acute capsaicin were not altered by endothelial damage, suggesting that substance P does not mediate the relaxation response to acute capsaicin administration. Exposure to capsaicin in vitro did not affect the neurogenic vasodilator response of cat cerebral arteries and did not alter substance P levels. Therefore, it was concluded that the acute effect of capsaicin is composed of two components. A contractile response is most likely due to direct effects on vascular smooth muscle, while a relaxation response is attributed to release of an as yet unidentified bioactive substance distinct from substance P.