Opiates, like other addictive drugs, elevate forebrain dopamine levels and are thought to do so mainly by inhibiting GABA neurons near the ventral tegmental area (VTA), in turn leading to a disinhibition of dopamine neurons. However, cholinergic inputs from the laterodorsal (LDT) and pedunculopontine (PPT) tegmental nucleus to the VTA and substantia nigra (SN) importantly contribute, as either LDT or PPT lesions strongly attenuate morphine-induced forebrain dopamine elevations. Pharmacological blockade of muscarinic acetylcholine receptors in the VTA or SN has similar effects. M 5 muscarinic receptors are the only muscarinic receptor subtype associated with VTA and SN dopamine neurons. Here we tested the contribution of M 5 muscarinic receptors to morphine-induced dopamine elevations by measuring nucleus accumbens dopamine efflux in response to intra-VTA morphine infusion using in vivo chronoamperometry. Intra-VTA morphine increased nucleus accumbens dopamine efflux in urethane-anesthetized wildtype mice starting at 10 min after infusion. These increases were absent in M 5 knockout mice and were similarly blocked by pre-treatment with VTA scopolamine in wildtype mice. Furthermore, in wildtype mice electrical stimulation of the PPT evoked an initial, short-lasting increase in striatal dopamine efflux, followed 5 min later by a second prolonged increase in dopamine efflux. In M 5 knockout mice, or following systemic pre-treatment with scopolamine in wildtype mice, the prolonged increase in striatal dopamine efflux was absent. The time course of increased accumbal dopamine efflux in wildtype mice following VTA morphine was consistent with both the prolonged M 5-mediated excitation of striatal dopamine efflux following PPT electrical stimulation and accumbal dopamine efflux following LDT electrical stimulation. Therefore, M 5 receptors appear critical for prolonged PPT excitation of dopamine efflux and for dopamine efflux induced by intra-VTA morphine.