[3H]Telenzepine has been shown to bind with high affinity (3 x 10(9) M-1) to a subpopulation of muscarinic binding sites in rat cerebral cortex, which have a high affinity for pirenzepine. The binding kinetics were very slow at 30 degrees. Only 50% of the [3H] telenzepine was found to be capable of binding to the receptors with high affinity. This suggested the presence of optical isomers of telenzepine. These were partially resolved on the picomole scale by using cortical muscarinic receptors to selectively bind the active isomer. It was then possible to measure the temperature and time dependence of the racemization of the inactive to the active enantiomer. The energy barrier for the inversion was 35 kcal/mol, and racemization was very slow even at 90 degrees. The affinity and selectivity of the unlabeled enantiomers for the different muscarinic receptor subtypes present on membranes from rat cerebral cortex, heart, and lacrimal gland was measured. The selectivity of active (+)-isomer was considerably greater than that of the (-)-isomer. As a consequence, the stereoselectivity of the enantiomers varied from 500 (M1 receptors in cerebral cortex) to 75 (cardiac receptors).