The activity of pancuronium as a muscarinic antagonist was compared in atria from several species and in guinea-pig ileal longitudinal muscle. Pancuronium had a 20-fold greater affinity for muscarinic receptors mediating the negative inotropic response to carbachol (CCh) in the guinea-pig left atrium (pKB = 6.96) than for muscarinic receptors in guinea-pig ileal longitudinal muscle (pKB = 5.65). The greater affinity of pancuronium in the atria was not due to an interaction with noradrenergic neurones as pretreatment of atria with propranolol (3 microM) or of guinea-pigs with 6-hydroxydopamine (400 mg/kg total dose) failed to modify the degree of inhibition exhibited in atria. The affinities of pancuronium for muscarinic receptors mediating negative inotropic responses in the kitten left atrium or negative chronotropic responses in guinea-pig or rabbit right atria were not significantly different from that in the guinea-pig left atrium (P greater than 0.05) but the affinity of pancuronium for muscarinic receptors in ileal longitudinal muscle was significantly different (P less than 0.05) from all values obtained in the cardiac preparations suggesting differences in the binding of the antagonist in smooth and cardiac muscle. The A-S plot for pancuronium (0.27 microM-0.20 mM) as an antagonist of negative inotropic responses to CCH was non-linear, showing a flattening at the higher concentrations of pancuronium. This was not apparent in the A-S plot for the inhibition of tonic responses to CCh in ileal longitudinal muscle.