Dissociation constants (KA) and relative efficacies of acetylcholine, (+)-methacholine and (-)-methacholine at muscarinic receptors in the guinea-pig isolated ileum were determined in the absence and presence of the cholinesterase inhibitor diisopropylfluorophosphate. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. The KA values, estimated after cholinesterase inhibition, of acetylcholine, (+)- and (-)-methacholine were 1.7, 2.0 and 620 microM, respectively. The large (730 fold) difference in spasmogenic activity between (+)- and (-)-methacholine is due primarily to a difference in affinity for ileal muscarinic receptors although differences in efficacy (2 to 4 fold) also contribute. It is suggested that the methyl group at the chiral centre of (+)-methacholine has no apparent effect on the binding to muscarinic receptors, whereas the corresponding methyl group of (-)-methacholine interferes with binding, presumably by stabilizing a conformation of the drug which does not fit the receptor very well.