Abstract 1. 1. The effect of chronic (19-day) treatment with the benzodiazepine tranquilizers diazepam (1 mg/kg/day, i.p.) and medazepam (5 mg/kg/day, i.p.) on the binding characteristics of muscarinic receptors in four rat brain structures: cerebral cortex, striatum, hippocampus and hypothalamus were studied using [ 3H]l-quinuclidinyl benzylate ([ 3H]QNB) as radioligand. 2. 2. Diazepam and medazepam treatment caused an overall decrease in muscarinic receptor binding affinity ( K d ). 3. 3. The number ( B max) of muscarinic receptors declined in the hippocampus and striatum and rose in the hypothalamus after both benzodiazepines. The B max of muscarinic receptors in the cerebral cortex was increased after diazepam treatment. 4. 4. The changes in the binding characteristics of muscarinic receptors might be due to benzodiazepine-induced occurrence of two populations of muscarinic binding sites: P 1 (high affinity, low capacity) and P 2 (low affinity, high capacity). 5. 5. The altered brain muscarinic receptor functions after chronic diazepam- or medazepam treatment suggest the role of cholinergic neurotransmission in the mechanism of action of benzodiazepines.