Muscarinic autoregulation of extracellular acetylcholine levels was investigated by microdialysis in the cerebral cortex of freely moving rats under basal conditions as well as following systemic administration of a reversible cholinesterase inhibitor. Atropine (2.2 mg/kg s.c. or 0.2 microM via the dialysis probe) did not affect basal extracellular acetylcholine levels in the cerebral cortex. However, it did potentiate the elevation of extracellular acetylcholine levels produced by a dose of systemic heptylphysostigmine which inhibited 25% of cortical and 40% of plasma cholinesterase activity. These observations suggest that the extracellular concentration of acetylcholine following moderate acetylcholinesterase inhibition is regulated through muscarinic receptors.