Until recently, little was known about the possible physiological functions of the M(5) muscarinic acetylcholine receptor subtype, the last member of the muscarinic receptor family (M(1)-M(5)) to be cloned. To learn more about the potential physiological roles of this receptor subtype, we generated and analyzed M(5) receptor-deficient mice (M5 -/- mice). Strikingly, acetylcholine, a potent dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5 -/- mice, suggesting that endothelial M(5) receptors mediate this activity in wild-type mice. This effect was specific for cerebral blood vessels, since acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5 -/- mice. In addition, in vitro neurotransmitter release experiments indicated that M(5) receptors located on dopaminergic nerve terminals play a role in facilitating muscarinic agonist-induced dopamine release in the striatum, consistent with the observation that the dopaminergic neurons innervating the striatum almost exclusively express the M(5) receptor subtype. We also found that the rewarding effects of morphine, the prototypical opiate analgesic, were substantially reduced in M5 -/- mice, as measured in the conditioned place preference paradigm. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5 -/- mice. It is likely that these behavioral deficits are caused by the lack of mesolimbic M(5) receptors, activation of which is known to stimulate dopamine release in the nucleus accumbens. These results convincingly demonstrate that the M(5) muscarinic receptor is involved in modulating several important pharmacological and behavioral functions. These findings may lead to novel therapeutic strategies for the treatment of drug addiction and certain cerebrovascular disorders.