Rat myocardial cells in tissue culture were utilized to investigate modulation by corticosteroids on muscarinic receptor affinity. Competition binding experiments between tritiated quinuclidinyl benzilate and carbachol were performed on the muscarinic receptor both on whole cells and on a crude membrane preparation from cultured myocardial cells. Hydrocortisone was shown to promote a transition of the high affinity site of the muscarinic receptor towards super-high affinity. This was analogous with the effect of divalent ions. The affinity shift was detectable after 6 hr exposure to 0.1 microgram/ml of hydrocortisone in the tissue culture dishes but was abolished by concomitant incubation with a protein synthesis inhibitor (10 microM puromycin). The response on agonist binding of Gpp(NH)p in the presence of manganese ions was altered indicating that the adenylate cyclase-coupled muscarinic receptor nucleotide binding protein was modulated. No effects of corticosteroid exposure were noted on phosphatidyl inositol turnover.