The second messenger response of cultured smooth muscle and endothelial cells of the rabbit aorta to acetylcholine (ACh) was investigated. ACh induced a concentration-dependent accumulation of inositol-monophosphate (InsP1) in the smooth muscle cells and a concentration-dependent reduction in basal production of adenosine 3',5'-cyclic monophosphate (cAMP) in the endothelial cells. Atropine, scopolamine and nitric oxide (NO) inhibited the ACh-induced accumulation of InsP1. The IC50 values were 55 +/- 4.3 nM, 81.2 +/- 6.5 microM and 13.3 +/- 3.5 microM, respectively. On the other hand, the inhibition of reduction in basal production of cAMP was inhibited by scopolamine (IC50 = 55.3 +/- 4.3 nM) and atropine (IC50 = 63.2 +/- 5.2 microM). Pirenzepine inhibited both the ACh-induced accumulation of InsP1 (IC50 = 1.5 +/- 0.01 microM) and reduction of basal production of cAMP (IC50 = 812 +/- 33.5 nM). However, unlike scopolamine or atropine, the M1-selective ligand was not selective to either the endothelial or smooth muscle receptors. These results demonstrate for the first time the second messenger response of the action of ACh on the endothelial muscarinic receptors and the inhibition of InsP1 formation by NO. In addition, the results also support our earlier findings that the muscarinic receptors in the endothelium and smooth muscle of the rabbit aorta can be differentiated by atropine and scopolamine, namely, the endothelial receptors have high affinity for scopolamine but extremely low affinity for atropine whilst the reverse holds true for the smooth muscle receptors.