1. The metabolism of phosphatidylinositol and phosphatidate was investigated in fragments of longitudinal smooth muscle from guinea-pig ileum incubated with cholinergic and anticholinergic drugs. 2. Incorporation of Pi into these lipids was enhanced by acetylcholine and carbamoylcholine. 3. The receptor responsible for triggering this response was of the muscarinic type, since (a) the response was also produced by the muscarinic agonists acetyl-beta-methylcholine, carbamoyl-beta-methylcholine and pilocarpine, and (b) the response was prevented by atropine and prophylbenzilylcholine mustard, but not by tubocurarine. 4. Increased phosphatidylinositol labellin was clearly observed within 5 min in tissue treated with a high concentration of carbamoylcholine. 5. Halfmaximal stimulation of phosphatidylinositol labelling occurred at approx. 10 muM-muM-carbamoylcholine. 6. Incubation of muscle fragments with carbamoylcholine provoked a decrease in phosphatidylinositol concentration, as would be expected if phosphatidyl-inositol breakdown is the reaction controlled by agonists. 7. This information all appears consistent with the proposal that phosphatidylinositol breakdown may be a reaction intrinsic to the mechanisms of muscarinic cholinergic receptor systems.