In incubations of dissociated adult rat anterior pituitary cells with [32P]orthophosphate, carbamylcholine causes an increase in phosphatidic acid labeling accompanied by a small reduction in phosphatidylinositol labeling. Carbamylcholine and oxotremorine produce about the same maximum change while that caused by pilocarpine is smaller. Low concentrations of atropine, quinuclidinyl benzilate, and scopolamine completely inhibit the effect caused by carbamylcholine, whereas d-tubocurarine does not decrease the stimulation, even at higher concentrations. The muscarinic antagonists alone produce a rise in phosphatidylinositol labeling and a drop in phosphatidic acid labeling, an effect opposite from that produced by the agonists, but d-tubocurarine alone has no effect. Thus changes in phospholipid metabolism are mediated through muscarinic cholinergic receptors in dissociated rat anterior pituitary cells, confirming the presence of functional binding sites. The present studies also demonstrate the utility of experiments on precursor incorporation into phospholipids in identifying the existence on cells or tissues of certain receptor classes.