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The present study aimed to directly characterize specific binding sites of tritium ([(3)H])-labeled imidafenacin, a new radioligand for labeling muscarinic receptors, in the bladder and other peripheral or central nervous tissues of rats. Muscarinic receptors in rat tissues were measured by radioligand binding assay using [(3)H]imidafenacin. Specific [(3)H]imidafenacin binding in rat tissues was saturable, reversible, and of high affinity. Estimated dissociation constants (Kd values) were significantly lower in submaxillary gland and prostate and higher in heart than in bladder, indicating lower Kd values in M1 and M3 subtype- than M2 subtype-dominating tissues. Unlabeled imidafenacin and clinically used antimuscarinic agents competed with [(3)H]imidafenacin for binding sites in bladder and other tissues in a concentration-dependent manner, which indicated pharmacological specificity of [(3)H]imidafenacin binding sites. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), an irreversible inactivating agent of M3 subtype, significantly decreased the number of [(3)H]imidafenacin binding sites in bladder, submaxillary gland, and colon, but not in heart. [(3)H]imidafenacin labeled muscarinic receptors in M1 and M3 subtype-dominating tissues with higher affinity than [N-methyl-(3)H]scopolamine methyl chloride (NMS). [(3)H]imidafenacin is a useful radioligand to label muscarinic receptors in M1- and M3-dominating tissues with high affinity.