Abstract 1. 1. Glycopyrrolate, a synthetic quaternary ammonium compound had biphasic effects on the contraction of guinea-pig atrium. At concentrations between 0.4 and 20 μM, glycopyrrolate induced a small but consistent increase in the contraction force. 2. 2. Further increase in the concentration of glycopyrrolate produced a concentration-dependent reduction in the force of contraction with a EC 50 of 0.24 mM. 3. 3. This negative inotropic effect was opposed by a Ca 2+ channel agonist, Bay K 8644. Glycopyrrolate also antagonized potently the depressant effects of carbachol and acetylcholine in guinea-pig atrium. 4. 4. Schild analysis showed a pA 2 value of 8.16 against carbachol and 8.39 against acetylcholine. These similar pA 2 values suggested that both compounds may have a common interacting site. The interactions however cannot be explained by a simple competition model as the slopes of the Schild plots were larger than unity. 5. 5. The mutual competition between glycopyrrolate and quinuclidinyl benzilate (QNB), gallamine or methoctramine indicated that glycopyrrolate could have multiple action sites in the atrium. Interaction at an allosteric site was implicated. 6. 6. Radioligand binding studies showed that glycopyrrolate displaced pirenzepine (PZ) and AF-DX 384 from their binding to the M 1 and M 2 muscarinic receptors in guinea-pig brain membranes respectively. 7. 7. The respective binding constants ( K is) were 0.60 and 0.03 nM. The Hill coefficient value ( n H) for glycopyrrolate against [ 3H] PZ was larger than unity, suggesting positive cooperativity at the receptor complex. In contrast, the n H of the agonist [ 3H] AF-DX 384 was not different from unity, indicating a simple competitive inhibition. 8. 8. Our results confirm the antimuscarinic properties of glycopyrrolate. We suggest that the therapeutic value of glycopyrrolate in the treatment of organophosphate poisoning is associated with its potency against prolonged muscarinic receptor activation by cholinergic agonists.