Despite the discovery of ion channels that are activated by protons, we still know relatively little about the signaling of acid pain. We used a novel technique, iontophoresis of protons, to investigate acid-induced pain in human volunteers. We found that transdermal iontophoresis of protons consistently caused moderate pain that was dose-dependent. A marked desensitization occurred with persistent stimulation, with a time constant of approximately 3 min. Recovery from desensitization occurred slowly, over many hours. Acid-induced pain was significantly augmented in skin sensitized by acute topical application of capsaicin. However, skin desensitized by repeated capsaicin application showed no significant reduction in acid-induced pain, suggesting that both capsaicin-sensitive and insensitive sensory neurons contribute to acid pain. Furthermore, topical application of non-steroidal anti-inflammatory drugs (NSAIDs) significantly attenuated acid-evoked pain but did not affect the heat pain threshold, suggesting a specific interaction between NSAIDs and peripheral acid sensors. Subcutaneous injection of amiloride (1 mm) also significantly inhibited the pain induced by iontophoresis of acid, suggesting an involvement of acid-sensing ion channel (ASIC) receptors. Conversely, iontophoresis of acid over a wide range of skin temperatures from 4 to 40 degrees C produced only minor changes in the induced pain. Together these data suggest a prominent role for ASIC channels and only a minor role for transient receptor potential vanilloid receptor-1 as mediators of cutaneous acid-induced pain.