A 30-min exposure to intact biting flies (stable flies) induced an opioid-mediated analgesia in fly-naive male deer mice, whereas exposure to either altered biting flies whose biting mouthparts were removed or nonbiting house flies had no significant effects. However, mice that were previously exposed to intact stable flies for 30 min exhibited significant analgesia when exposed 24-168 hr later to stable flies whose biting parts were removed, but not to nonbiting house flies. Administration of the specific N-methyl-D-aspartate (NMDA) antagonist NPC 12626 to fly-naive mice before exposure to intact flies, although not significantly reducing the analgesic response, blocked the subsequent conditioned analgesia. Naloxone, which blocked the intact biting fly-induced analgesia, did not alter the acquisition of the conditioned analgesic response to the altered stable flies. This demonstrates an NMDA-mediated acquisition of conditioned analgesia to a natural aversive stimulus.