Opioid-induced respiratory depression is well documented. However, exact sites of action and mechanisms for opioid-induced effects on respiration have not yet been elucidated. The present study was carried out on isolated brainstem–spinal cord preparations from newborn rats in order to explore the opioid activity on brainstem μ-, δ- and κ-receptors. The brainstem–spinal cord was isolated from 0- to 4-day-old Sprague-Dawley rats. The preparation was perfused with artificial cerebrospinal fluid (28.5°C) equilibrated with 95% O₂ and 5% CO₂ at a pH of 7.4. Neuronal respiratory activity was recorded from the ventrolateral part of the medulla oblongata and efferent impulses from C4 or C5 ventral roots. Effects of the μ-receptor agonist DAGO, the δ-receptor agonist DPDPE and the κ-receptor agonist U50,488 on respiratory frequency (f_R), inspiratory time (T_i) and peak integrated C4 amplitude (Int_C4) were measured. In addition, the effect of pre-treatment with the μ₁ receptor antagonist naloxanazine (35 mg/kg, subcutaneous injection) was evaluated. DAGO reduced f_R and T_i in a concentration-dependent manner and caused a reduction of Int_C4 at high concentrations (10 μM). The μ₁ receptor antagonist naloxanazine shifted the f_R concentration–response curve for DAGO to the right (P<0.05). DPDPE had no effect on respiratory activities whereas U50,488, like DAGO, reduced f_R and Int_C4 in a concentration-dependent manner. It was concluded that μ-opioid receptors, including the μ₁ were involved in f_R reduction whereas κ-opioid receptors were involved in reduction of both f_R and respiratory amplitude. δ-Opioid receptors do not seem to participate in respiratory modulation at this age.