Repeated microinjections of morphine into the ventrolateral periaqueductal gray produce antinociceptive tolerance. This tolerance may be a direct effect of morphine on cells within the ventrolateral periaqueductal gray or may require activation of downstream structures such as the rostral ventromedial medulla or spinal cord. Experiment 1 examined whether tolerance develops when opioid receptors in the ventrolateral periaqueductal gray are blocked prior to repeated systemic morphine administration. Microinjections of naltrexone hydrochloride (1microg/0.4microl) into the ventrolateral periaqueductal gray blocked antinociception and significantly attenuated the development of antinociceptive tolerance produced from systemic morphine administration. Experiment 2 examined whether tolerance develops when the effects of morphine are isolated to the ventrolateral periaqueductal gray. This was accomplished by microinjecting morphine (5microg/0.4microl) into the ventrolateral periaqueductal gray while simultaneously blocking the descending output through the rostral ventromedial medulla. Inhibition of neurons within the rostral ventromedial medulla by microinjecting the GABA(A) agonist muscimol (10ng/0.5microl) blocked the antinociception produced by microinjection of morphine into the ventrolateral periaqueductal gray but did not block the development of tolerance. These data demonstrate that the ventrolateral periaqueductal gray is both necessary and sufficient to produce tolerance to the antinociceptive effect of morphine. The ventrolateral periaqueductal gray is necessary in that tolerance does not develop if opiate action within the ventrolateral periaqueductal gray is blocked (experiment 1). The ventrolateral periaqueductal gray is sufficient in that tolerance occurs even when morphine's effects are restricted to the ventrolateral periaqueductal gray (experiment 2).