The novel quaternary ammonium salt (3R)-3- [[[(3-fluorophenyl) [(3,4,5-trifluorophenyl) H]tiotropium dissociated slowly from both hM3 (t 1 ⁄2 ϭ 163 min) and hM2 receptor (t 1 ⁄2 ϭ 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC 50 ϭ 9.0 -9.6) and longlasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED 50 value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3