1. An intrathecal (i.t.) injection of a selective delta 2-opioid receptor agonist, [D-Ala2]deltorphin II, produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala2]deltorphin II. This acute tolerance lasted 3 to 9 h and completely subsided by 12 h. The experiments were designed to examine the effect of pretreatment with an antisense oligodeoxynucleotide to delta 2-opioid receptor mRNA (delta-AS oligo) on the recovery from tolerance to [D-Ala2]deltorphin II-induced antinociception in male ICR mice. 2. Pretreatment with delta-AS oligo (1.63 to 163 pmol, i.t.), but not mismatched oligo (MM oligo) (163 pmol), prevented the recovery from acute tolerance to [D-Ala2]deltorphin II-induced antinociception in a dose-dependent manner. However, treatment with delta-AS oligo (163 pmol) did not prevent the recovery from tolerance to either the mu-opioid receptor agonist [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO) or the kappa-opioid receptor agonist U50,488H, indicating subtype specificity in the mechanism by which delta-AS oligo inhibits recovery from delta 2-opioid tolerance. 3. Treatment with [D-Ala2]deltorphin II (i.t.) significantly reduced the binding of [tyrosyl-3,5-(3)H(N)]-Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]-DSLET), a delta 2-opioid receptor agonist ligand, in the spinal cord 3 h after treatment, but binding returned to control levels by 24 h after treatment. However, [3H]-DSLET binding in the spinal cord remained significantly reduced at 24 h if delta-AS oligo (163 pmol) was coadministered with [D-Ala2]deltorphin II (6.4 nmol). 4. Based on these findings, it is concluded that a single stimulation of spinal cord delta 2-opioid receptors by intrathecally-administered [D-Ala2]deltorphin II induces a long-lasting desensitization of delta 2-opioid receptors to [D-Ala2]deltorphin II. Recovery from delta 2-opioid receptor-mediated antinociceptive tolerance apparently depends on replenishment by newly synthesized delta 2-opioid receptor protein rather than immediate reversal of delta 2-opioid receptors.