Displacement of [3H]oxotremorine-M [( 3H]oxo-M) binding by muscarinic antagonists that are functionally non-selective (atropine), ileoselective (4-DAMP) or cardioselective (gallamine, pancuronium, vecuronium, himbacine) was investigated in guinea-pig atrial and ileal longitudinal muscle membranes. [3H]Oxo-M bound to a single population of high affinity sites in atrial (KD = 11.40 nM) and ileal (KD = 6.15 nM) membranes. Atropine displaced [3H]oxo-M binding sites in a competitive manner, showing similar affinities in the two tissues. 4-DAMP showed two binding sites in ileum but not in atria. The dissociation constant at the high affinity site in ileum was ca 5-fold lower than the value observed in atria, indicating ileoselectivity. Vecuronium also displaced [3H]oxo-M binding in a competitive manner and exhibited similar affinities in both tissues. Gallamine, pancuronium and himbacine displayed two binding sites in each of the two tissues with the majority of sites (ca. 60-80%) showing high affinity. Overall the cardioselective antagonists do not exhibit any consistent correlation between the affinities found in functional experiments and those determined in binding experiments.