Background: We previously reported that an 18–24 h pretreatment of mice with Gi2α G-protein subunit antisense oligonucleotide (oligo) attenuates the supraspinal (intracerebroventricular; i.c.v.) μ-opioid-induced antinociception in the tail-immersion test (55°C). Purpose: To measure the time course of effect of Gi2α antisense on i. c. v. μ-opioid-induced antinociception. Methods: Mice were injected i. c. v. with 33-mer oligodeoxyribonucleotide (6.0 nmol) directed against Gi1α or Gi2α or with vehicle and the antinociceptive response to i. c. v. μ-opioid agonist (morphine, [D-Ala2,NMePhe4, Gly5-ol]enkephalin (DAMGO) or sufentanil) was determined 0.75 h to 8 d later using the tail-immersion test. Results: Gi2α antisense, but not control Gi1α antisense, attenuated antinociception in the order: morphine > DAMGO > sufentanil (the inverse order of their intrinsic efficacy) throughout the entire time-course of the antisense effect. The on-set of antisense oligo effect was about 0.75 h and the effect lasted up to 48 h (for morphine). Conclusions: The rapidity of the onset of antisense oligo effect suggests a rapid turnover of a subunits of α-proteins coupled to μ-opioid receptors. In addition, the data support our previous suggestion that opioid antinociceptive efficacy is inversely related to sensitivity to G-protein oligo antisense.