Activation of muscarinic acetylcholine receptors is primarily responsible for urinary bladder emptying. Because multiple subtypes of muscarinic receptors exist, we wished to characterize those present in bladder and ultimately to attribute function to those that regulate bladder contractility, neurotransmitter release and perhaps other cholinergic functions in this tissue. Although the m2 and m3 subtypes could be immunoprecipitated after solubilization from human, rat, rabbit and guinea pig bladder membranes, the m1, m4 and m5 subtypes could not. The m2:m3 ratio was 9:1 in rat bladder but was only 3:1 in the other species examined. Immunoprecipitation of the m2 subtype correlated with the relative levels of high-affinity agonist binding sites measured by competition of carbachol for [3H]N-methylscopolamine binding or measured directly using [3H]oxotremorine-M. In the presence of agonist, but not antagonist, GTP binding proteins could be immunoprecipitated in concert with the m2 or m3 receptors using anti-receptor antibodies. These proteins were members of the Gi and Gq/11 subfamilies for both the m2 and the m3 receptor subtypes. In spite of the preponderance of the m2 receptor in all species studied, Schild analysis using somewhat selective antagonists showed that the pharmacologically defined m3 receptor mediated contractility in strips of rat and rabbit bladder. Thus acetylcholine activates bladder smooth muscle via the m3 receptor subtype, and subsequent contractility may be transduced by guanine nucleotide binding proteins such as the Gi and Gq/11 subfamilies.