cis-Methyldioxolane (CD) is a muscarinic receptor agonist. [3H]CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [3H]CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [3H]CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [3H]CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [3H]CD with a KD of 3.5 +/- 0.5 nM. Binding was eliminated in the presence of guanosine 5'-O-(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [3H]CD are those that are associated with GDP-bound G protein. Binding of [3H]CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [3H]CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [3H]CD should aid research in these areas.