Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.