Muscarinic acetylcholine M2 and M3 receptor subtypes are coexpressed in many types of smooth muscle including gastrointestinal smooth muscle, urinary bladder and vascular and airway tissue. Activation of M3 receptors, via the G protein Gq, results in increased polyphosphoinositide hydrolysis, release of Ca2+ ions from the sarcoplasmic reticulum and consequently causes contraction. Quantitation of the relative expression of M2 and M3 receptors has shown that the proportion of M2 receptors often predominates over the M3 receptor population by 4:1 or more. Although it is established that M2 receptors preferentially link, via a pertussis-toxin-sensitive G protein Gi, to inhibition of adenylate cyclase activity, relatively little is known concerning the physiological role of the M2 receptor population. In this review, Richard Eglen and colleagues discuss recent data concerning the possible role(s) of muscarinic receptor subtypes in smooth muscle and appraise the pharmacological methods for dissecting the function of muscarinic receptor subtypes in tissues co-expressing multiple receptors.