Rat thymocytes possess a single class of saturable, high affinity binding sites for muscarinic antagonists of the benzilate type such as [3H]3-quinuclidinyl benzilate ([3H]3-QNB). The average number of receptors per cell is 3000 and the equilibrium dissociation constant of [3H]3-QNB on intact cells is 7.5 nM. In the work reported here we found that perturbation of the thymocyte membrane by addition of phytohemagglutinin (4 micrograms/ml) caused a transient increase in muscarinic antagonist binding, and hydrocortisone (100 mg/kg s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of muscarinic receptor sites on thymocytes by 100%. Atropine treatment, which in other tissues causes increased muscarinic receptor concentration, did not alter the receptor number on thymocytes. Binding of carbachol to the receptor on intact cells resulted in inhibition of cAMP synthesis and stimulation of cGMP synthesis. These muscarinic agonist effects were each inhibited by the simultaneous addition of the muscarinic antagonist atropine (5 X 10(-5) M). No stimulation of phosphatidylinositol turnover by muscarinic agonists was observed.