In this report, muscarinic receptor-mediated phosphoinositide (PI) hydrolysis is characterized pharmacologically in the rat retina. In the presence of eserine, acetylcholine (ACh) elicited a concentration-dependent increase in inositol monophosphate with a calculated EC50 of about 2.8 microM. Maximum increase was achieved with about 100 microM ACh. Cholinergic receptor agonists stimulated phospholipase C-mediated hydrolysis of PI with the following rank order of potency: ACh = oxotremorine greater than McN-A-343 greater than bethanechol greater than arecoline = carbachol greater than muscarine. Oxotremorine analogs stimulated PI hydrolysis with the following rank order of potency: ACh = oxotremorine = oxotremorine-2 greater than oxotremorine-M = oxotremorine-4. Carbachol-mediated Pl hydrolysis was blocked by atropine and by the putatively selective muscarinic type 1 (M1) receptor antagonist, pirenzepine, with apparent Ki values of 0.1 and 1.0 nM, respectively. In contrast, the selective muscarinic type 2 (M2) antagonists, gallamine and AF-DX 116, failed to inhibit the action of carbachol. These findings demonstrate that stimulation of muscarinic receptors in the rat retina leads to PI hydrolysis and that these receptors appear to be M1 cholinergic receptors.