The effects of three concentrations of aqueous-ethanolic extract, 10 nM atropine and saline on muscarinic receptors were tested on two groups of non-incubated (group 1; n = 7) and incubated tracheal chains with propranolol and chlorpheniramine (group 2; n = 6). The EC₅₀ of higher concentration of the extract (2 µg mL⁻¹) in group 2 was significantly greater than those of group 1 (p < 0.01). The maximum response obtained in the presence of all concentrations of extract in groups 1 and 2 were lower than saline (p < 0.05 to 0.001). There were parallel rightward shifts in the concentration response curves obtained for all concentrations of the extract in group 2. There was significant and positive correlation between the concentrations and the values of EC₅₀ in group 1 (p < 0.05) and group 2 (p < 0.001). These results indicate an anti-cholinergic effect of Zataria multiflora Boiss on the tracheal chains of guinea pigs. A β-adrenoceptor stimulatory effect for the plant was also suggested.