(D-ALa2, NMePhe4, Gly-ol5) encephalin (DAMGO), a selective mu-opioid receptor agonist, has previously been demonstrated to inhibit the cholinergic and the noncholinergic contraction in guinea-pig airways. In contrast, opioids had no inhibitory effect on cholinergic neurotransmission in the upper trachea when stimulated at 8 Hz. We investigated whether DAMGO, a selective mu-opioid receptor agonist, [D-Pen 2,5] encephalin (DPDPE), a selective delta-opioid receptor agonist, and U-69593, a selective kappa-opioid receptor agonist could modulate the cholinergic contraction in the upper trachea at different frequencies of stimulation. Moreover, we have investigated whether DAMGO, DPDPE and U-69593 could also modulate the iNANC relaxation. DAMGO (1-100 microM) inhibited the cholinergic contraction in the upper trachea with a maximum inhibition of 57+/-15% at 1 Hz (n=4; p<0.05). On the other hand, DPDPE (10 microM) and U69593 (10 microM) did not produce any significant inhibition of the cholinergic contraction. Naloxone, an opioid receptor antagonist (100 microM), was able to antagonize the inhibitory effect of DAMGO (n=5; p<0.01) on the cholinergic contraction at a frequency of 2 Hz. DAMGO (10 microM) did not displace the cumulative concentration-response relationship to acetylcholine (10 nM-10 mM), (n=4; NS). This provides evidence that prejunctional mu-opioid receptors (and not delta-opioid or kappa-opioid receptors) modulate cholinergic contraction in the upper trachea. In contrast, DAMGO (10 microM) had no significant inhibitory effect on the nonadrenergic relaxation (n=4; NS) in the upper trachea. Neither DPDPE nor U-69593 had any effect on the nonadrenergic relaxation. These findings suggest that DAMGO directly inhibits the cholinergic contraction and that the opioid receptor involved in the inhibition of the cholinergic contraction in the upper trachea is of the mu-opioid type. The finding that opioids inhibit cholinergic contraction without altering NANC relaxations suggests that distinct populations of nerves mediate these two effects.