The neuromuscular blocking drug alcuronium was found earlier to increase the affinity of muscarinic receptors for methyl-N-scopolamine (NMS). This effect could be observed in some but not in other tissues. Subtype selectivity of the positive allosteric action of alcuronium was now investigated in radioligand binding experiments in Chinese hamster ovary (CHO) cells stably transfected with the genes encoding the M1-M5 subtypes of muscarinic receptors. Alcuronium had a particularly high affinity for the M2 receptor subtype (Kd = 0.6 microM) and its affinity for muscarinic receptor subtypes diminished in the order M2 > M4 = M3 > M1 > M5. Alcuronium allosterically increased the binding of (3H)NMS to membranes containing receptors of the M2 (cooperativity factor alpha = 0.38) and M4 subtypes (alpha = 0.72) and inhibited the binding of (3H)NMS to membranes containing receptors of the M1, M3 and M5 subtypes (alpha = 3.35-4.35). The positive effects of alcuronium could also be observed in experiments with (3H)NMS binding to intact whole cells, indicating that the positive allosteric action of alcuronium occurs by alcuronium binding to receptor domains that are accessible from the extracellular space. Alcuronium diminished the affinity for (3H)quinuclidinyl benzilate [(3H)QNB] at all five subtypes of muscarinic receptors and slowed down the dissociation of both (3H)NMS and (3H)QNB; its decelerating effect on radioligand dissociation was most pronounced at the M2 receptor subtype. Differences between the effects of alcuronium on individual muscarinic receptor subtypes are apparently responsible for differences between the allosteric effects of alcuronium on muscarinic receptors in various tissues that had been described previously.