Abstract Endothelin-1 (ET-1) acts on endothelial cells to enhance mechanical stimulation–induced release of adenosine triphosphate (ATP), which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study, we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane–associated ATP synthase, ATP-binding cassette transporters, and ion channels. Inhibitors of vesicular exocytosis (ie, monensin, brefeldin A, and bafilomycin), plasma membrane–associated ATPase (ie, oligomycin and pigment epithelium–derived factor peptide 34-mer), and connexin ion channels (carbenoxolone and flufenamic acid) but not ATP-binding cassette transporter (ie, dipyridamole, nicardipine, or CFTRinh-172) attenuated SDH. This study reports a role of ATP in SDH and suggests novel targets for the treatment of vascular pain syndromes. Perspective ET-1 acts on endothelial cells to produce mechanical stimulation–induced hyperalgesia. Inhibitors of 3 different ATP release mechanisms attenuated this SDH. This study provides support for a role of ATP in SDH and suggests novel targets for the treatment of vascular pain syndromes.