Existing antimuscarinic drugs for overactive bladder have high affinity for M(3)/M(1) muscarinic receptors and consequently produce M(3)/M(1)-mediated adverse effects including dry mouth, constipation, mydriasis and somnolence. TD-6301 is a M(2/4) muscarinic receptor-selective antagonist developed for the treatment of overactive bladder. The present studies characterize the in vitro and in vivo pharmacological properties of this molecule in comparison to other marketed antimuscarinics agents. In radioligand binding studies, TD-6301 was found to possess high affinity for human M(2) muscarinic receptor (K(i)=0.36 nM) and was 31, 36, 2 and 128-fold selective for the human M(2) muscarinic receptor compared to the M(1), M(3), M(4) and M(5) muscarinic receptors, respectively. The in vivo bladder selectivity of TD-6301 in rats was determined to be 26, 28, >100, 16 and 0.4-fold, respectively, assessed by comparing its potency for inhibition of volume-induced bladder contractions to that for inhibition of oxotremorine-induced salivation, inhibition of small-intestinal transit, decreases in locomotor activity, increases in pupil diameter and increases in heart rate. TD-6301 was more potent in inhibiting volume-induced bladder contractions (ID(50)=0.075 mg/kg) compared to oxotremorine-induced salivation (ID(50)=1.0 mg/kg) resulting in a bladder/salivary gland selectivity ratio greater than that observed for tolterodine, oxybutynin, darifenacin and solifenacin. The preclinical properties of TD-6301 suggest that this molecule is likely to be efficacious in overactive bladder patients with a lower propensity to cause M(3) muscarinic receptor mediated adverse effects.