Learning processes such as respondent or Pavlovian conditioning are believed to play an important role in the development of chronic pain, however, their influence on the inhibition of pain has so far not been assessed in humans. The purpose of this study was the demonstration of Pavlovian conditioning of stress-induced analgesia in humans and the determination of its opioid mediation. In a differential classical conditioning paradigm two different auditory stimuli served as conditioned stimuli and mental arithmetic plus white noise as unconditioned stimulus. Subsequent to four conditioning trials naloxone or placebo was applied in a double-blind fashion on two test days. Both pain threshold and pain tolerance showed conditioned stress-induced analgesia. Pain tolerance was affected by naloxone whereas pain threshold was not. The data of this study show that stress analgesia can be conditioned in humans and that it is at least partially mediated by the endogenous opioid system. Learning processes also influence pain inhibitory processes in humans and this effect might play a role in the development of chronic pain.