The binding selectivity of the muscarinic antagonist tripitramine has been tested on the five cloned human muscarinic receptor subtypes (Hm1 to Hm5) expressed in chinese hamster ovary (CHO-K1) cells. The results indicate that tripitramine binds to the muscarinic Hm2 receptor with a Ki value of 0.27 +/- 0.02 nM. Tripitramine distinguishes Hm2 vs. Hm4 by a factor of 24 and vs. Hm3 and Hm5 by a factor of 142 and 125, respectively. A lower affinity ratio, about 6-fold, was found between muscarinic Hm2 and Hm1 receptors. A comparative study with the well-known selective muscarinic M2 receptor antagonist methoctramine indicates that tripitramine has gained both potency and selectivity for the muscarinic Hm2 receptor subtype.