The type-5 muscarinic acetylcholine receptor (mAChR, M5) is almost exclusively expressed in dopamine (DA) neurons of the ventral tegmental area and substantia nigra pars compacta; therefore, they are ideally located to modulate DA signaling and underlying behaviors. However, the role of M5 in shaping DA release is still poorly characterized. In this study, we first quantitatively mapped the expression of M5 in different neurons of the mouse midbrain, then used voltammetry in mouse striatum to evaluate the effect of M5-selective modulators on DA release. The M5 negative allosteric modulator ML375 significantly decreased electrically evoked DA release and blocked the effect of Oxotremorine-M (Oxo-M; nonselective mAChR agonist) on DA release in the presence of an acetylcholine nicotinic receptor blocker. Conversely, the M5 positive allosteric modulator VU 0365114 significantly increased electrically evoked DA release and the Oxo-M effect on DA release. We then assessed M5's impact on mesolimbic circuit function in vivo. Although psychostimulant-induced locomotor activity models in knockout mice have previously been used to characterize the role of M5 in DA transmission, the results of these studies conflict, leading us to select a different in vivo model, namely a cocaine self-administration paradigm. In contrast to a previous study that also used this model, in the current study, administration of ML375 did not decrease cocaine self-administration in rats (using fixed and progressive ratio). These conflicting results illustrate the complexity of M5 modulation and the need to further characterize its involvement in the regulation of dopamine signaling, central to multiple neuropsychiatric diseases. SIGNIFICANCE STATEMENT: This work describes the type-5 muscarinic receptor (M5) pattern of expression within the midbrain as well as its physiological modulation by selective compounds at the axon terminal level in the striatum, where M5 directly shapes dopamine transmission. It offers the first direct readout of mesolimbic dopamine release modulation by M5, highlighting its role in regulating neurocircuits implicated in the pathophysiology of neuropsychiatric disorders such as substance use disorders, major depressive disorder, and schizophrenia.