Opioids can modulate breathing and predispose to respiratory depression by actions at various central nervous system sites, but the mechanisms operating at respiratory motor nuclei have not been studied. This study tests the hypotheses that (i) local delivery of the μ-opioid receptor agonist fentanyl into the hypoglossal motor nucleus (HMN) will suppress genioglossus activity in vivo, (ii) a component of this suppression is mediated by opioid-induced acetylcholine release acting at muscarinic receptors, and (iii) δ- and κ-opioid receptors also modulate genioglossus activity. Seventy-two isoflurane-anaesthetised, tracheotomised, spontaneously breathing rats were studied during microdialysis perfusion into the HMN of (i) fentanyl and naloxone (μ-opioid receptor antagonist), (ii) fentanyl with and without co-application of muscarinic receptor antagonists, and (iii) δ- and κ-opioid receptor agonists and antagonists. The results showed (i) that fentanyl at the HMN caused a suppression of genioglossus activity (P < 0.001) that reversed with naloxone (P < 0.001), (ii) that neither atropine nor scopolamine affected the fentanyl-induced suppression of genioglossus activity, and (iii) that δ-, but not κ-, opioid receptor stimulation also suppressed genioglossus activity (P = 0.036 and P = 0.402 respectively). We conclude that μ-opioid receptor stimulation suppresses motor output from a central respiratory motoneuronal pool that activates genioglossus muscle, and this suppression does not involve muscarinic receptor-mediated inhibition. This μ-opioid receptor-induced suppression of tongue muscle activity by effects at the hypoglossal motor pool may underlie the clinical concern regarding adverse upper airway function with μ-opioid analgesics. The inhibitory effects of μ- and δ-opioid receptors at the HMN also indicate an influence of endogenous enkephalins and endorphins in respiratory motor control.