The effect of a long-term exposure (5 days) to atropine on muscarinic acetylcholine receptors and receptor-coupled second messenger systems was investigated using mouse cerebral cortical neurons in primary culture. The long-term exposure of neurons to atropine (10 nM) induced increases in both the Bmax and Kd values of [3H]quinuclidinyl benzilate (QNB) binding to muscarinic acetylcholine receptors. Alterations in muscarinic receptor-coupled second messenger systems, such as phosphoinositide (PI) hydrolysis and cyclic GMP (cGMP) formation following a long-term exposure to atropine, were also examined. Carbachol-stimulated PI hydrolysis was found to be decreased by the exposure to atropine in spite of the increase of muscarinic receptors. In addition, a long-term exposure to atropine had no effect on carbachol-stimulated cGMP formation as well as on the rightward shift of the carbachol competition curve of [3H]QNB binding in the presence of GTP. These results suggest that the up-regulation in muscarinic cholinergic receptors induced by long-term exposure to atropine may involve not only the increase in number of muscarinic receptors but also the decreased responsiveness in muscarinic receptor-coupled second messenger systems.