The affinity of amitriptyline for muscarinic receptors in rat brain areas was studied using autoradiographic techniques including image analysis. As shown by competitive inhibition of [(3)H]-l-quinuclidinyl benzilate binding, amitriptyline was found to be a potent inhibitor of muscarinic receptors throughout the rat brain. Muscarinic receptors in the external layers of the cortex displayed a high affinity for amitriptyline (IC(50) = 65.8 +/- 2.1 nM), while the hippocampal regions had somewhat lower affinities (e.g. IC(50) = 96.3 +/- 3.4 nM). Amitriptyline bound with lower affinity in the thalamus and various midbrain regions, such as the paraventricular nucleus of the thalamus and the superior colliculus, which had IC(50) values of 112 +/- 6.8 and 117 +/- 32.6 nM, respectively. Other midbrain regions displayed higher affinities, for example, the substantia nigra had an IC(50) value of 62.8 +/- 0.9 nM. The data show that amitriptyline binds with high affinity to muscarinic receptors with a modest subtype selectivity that is unlike that of either pirenzepine or AF-DX 116. In addition, amitriptyline at concentrations of 10 nM-1 ?M antagonized the oxotremorine-induced inhibition of acetylcholine release in cortical nerve endings, demonstrating activity at M(2) autoreceptors.