Background: Morphine and fentanyl are two of the most commonly used opioids to treat pain. Although both opioids produce antinociception by binding to mu-opioid receptors (MOR), they appear to act via distinct signalling pathways. Objective: This study will reveal whether differences in morphine and fentanyl antinociception are the result of selective activation of G-protein signalling and/or selective activation of pre- or postsynaptic MORs. Methods: The contribution of each mechanism to morphine and fentanyl antinociception was assessed by microinjecting drugs to alter G-protein signalling or block potassium channels linked to pre- and postsynaptic MORs in the ventrolateral periaqueductal gray (PAG) of male Sprague-Dawley rats. Results: Both morphine and fentanyl produced a dose-dependent antinociception when microinjected into the PAG. Enhancement of intracellular G-protein signalling by microinjection of the Regulator of G-protein Signalling 4 antagonist CCG-63802 into the PAG enhanced the antinociceptive potency of morphine, but not fentanyl. Microinjection of α-dendrotoxin into the PAG to block MOR activation of presynaptic Kv + channels caused a significant rightward shift in the dose–response curve of both morphine and fentanyl. Microinjection of tertiapin-Q to block MOR activation of postsynaptic GIRK channels caused a larger shift in the dose–response curve for fentanyl than morphine antinociception. Conclusions: These findings reveal different PAG signalling mechanisms for morphine and fentanyl antinociception. In contrast with fentanyl, the antinociceptive effects of morphine are mediated by G-protein signalling primarily activated by presynaptic MORs. Significance: Microinjection of the opioids morphine and fentanyl into the periaqueductal gray (PAG) produce antinociception via mu-opioid receptor signalling. This study reveals differences in the signalling mechanisms underlying morphine and fentanyl antinociception in the PAG. In contrast with fentanyl, morphine antinociception is primarily mediated by presynaptic opioid receptors and is enhanced by blocking RGS proteins.