An injection of acetylcholine (ACh) into renal artery is known to cause diuresis. In brain and other organs, cholinergic agents have been shown to produce their actions through the phosphoinositide (PI) second messenger system. To determine if cholinergic agents also produce activation of the PI messenger system in the kidney, we investigated the effects of carbachol (a stable analog of acetylcholine) on PI hydrolysis in the cortex, outer medulla and inner medulla of the rabbit kidney. PI hydrolysis was determined by measuring the formation of inositol phosphates in response to stimulation by carbachol in the presence of 8 mM lithium. Carbachol, 1 mM, was able to stimulate PI hydrolysis in the inner medulla and outer medulla (622 and 388% over control values, respectively), but not the cortex. The response to carbachol in the inner medulla was concentration-dependent (EC50 = 10(-5) M). The response was blocked by 1 microM atropine and not by 1 microM hexamethonium. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide did not stimulate PI hydrolysis. The effect of carbachol was dependent upon the presence of calcium ions. Substitution of alpha-ketoglutarate for glucose inhibited the response to carbachol in the inner medulla, suggesting a specific substrate requirement in PI metabolism. It is concluded that cholinergic agents produce stimulation of PI hydrolysis through muscarinic receptors in the inner medulla. Whether PI second messenger system in the kidney is involved in the diuretic effect of cholinergic agents remains to be determined.