Despite synergistic morbidity and mortality, concomitant consumption of alcohol and tobacco is increasing, and their antinociceptive effect has been linked with co-abuse. Present study was designed to investigate the role of imidazoline binding sites in the antinociceptive effect of nicotine, ethanol, and their combination. Separate group of male Sprague–Dawley rats (200–250 g) were treated with different doses of alcohol (0.50–2 g/kg, i.p.) or nicotine (0.25–1 mg/kg, i.p.), and their combination evaluated in tail flick test. Influence of endogenous imidazoline binding site ligands, agonist, and antagonists were determined by their prior treatment with effective or subeffective doses of either ethanol or nicotine. Ethanol, nicotine, or their subeffective dose combination exhibited significant antinociceptive effects in dose-dependent manner. Antinociceptive effect of ethanol and nicotine was significantly augmented by intracerebroventricular (i.c.v.) administration of endogenous imidazoline receptor ligands, harmane (25 μg/rat, i.c.v.) and agmatine (10 μg/rat, i.c.v.), as well as imidazoline I1/α2 adrenergic receptor agonist, clonidine (2 μg/rat, i.c.v.), I1 agonist moxonidine (25 μg/rat, i.c.v.), and imidazoline I2 agonist, 2-BFI (10 μg/rat, i.c.v.). Conversely, antinociception elicited by ethanol or nicotine or their subeffective dose combination was antagonized by pretreatment with imidazoline I1 antagonist, efaroxan (10 μg/rat, i.c.v.), and I2 antagonist, idazoxan (4 μg/rat, i.c.v.), at their per se ineffective doses. These findings project imidazoline binding ligands as important therapeutic molecules for central antinociceptive activity as well as may reduce the co-abuse potential of alcohol and nicotine.