The in vitro effect of various diuretics on rat kidney adenylate cyclase was investigated in crude homogenates of the cortex, the outer and inner medulla. 10-3 M furosemide inhibited adenylate cyclase by 40% in the cortex, by 16% in the outer medulla and by 43% in the inner medulla. 10-3 M ethacrynic acid inhibited adenylate cyclase activity by 65% in the cortex, 59% in the outer medulla and by 57% in the inner medulla. Amiloride produced no significant inhibition of the adenylate cyclase reaction. In the cortex, furosemide partially inhibited adenylate cyclase under basal, fluoride-stimulated and parathyroid hormone-stimulated conditions. Ethacrynic acid produced a strong inhibition of adenylate cyclase activation by F- and parathyroid hormone. In the inner medulla 10-2 M F- and 1 mU antidiuretic hormone reversed the furosemide effect on adenylate cyclase. Ethacrynic acid produced a strong inhibition of adenylate cyclase in the presence of F- and antidiuretic hormone. It is suggested that inhibition of renal adenylate cyclase might be a possible mode of action of certain diuretics.