A large body of evidence indicates important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the A_2A receptor gene have been developed successfully, and these animals were shown to be hypoalgesic. To investigate whether there are any compensatory alterations in opioid systems in mutant animals, we have performed quantitative autoradiographic mapping of μ, δ, κ, and opioid receptor-like (ORL1) opioid receptors in the brains and spinal cords of wild-type and homozygous A_2A receptor knock-out mice. In addition, μ-, δ-, and κ−mediated antinociception using the tail immersion test was tested in wild-type and homozygous A_2A receptor knock-out mice. A significant reduction in [³H]deltorphin-I binding to δ receptors and a significant increase in [³H]CI-977 binding to κ receptors was detected in the spinal cords but not in the brains of the knock-out mice. μ and ORL1 receptor expression were not altered significantly. Moreover, a significant reduction in δ-mediated antinociception and a significant increase in κ-mediated antinociception were detected in mutant mice, whereas μ-mediated antinociception was unaffected. Comparison of basal nociceptive latencies showed a significant hypoalgesia in knock-out mice when tested at 55°C but not at 52°C. The results suggest a functional interaction between the spinal δ and κ opioid and the peripheral adenosine system in the control of pain pathways.