Effects of parathyroid hormone (PTH), calcitonin (CT), vasopressin (VP), and glucagon (GL) on adenylate cyclase activity and cyclic AMP (cAMP) levels in isolated cortical thick ascending limbs of Henle's loop (CTAL) of the rat kidney were examined. PTH, CT, and VP each stimulated adenylate cyclase of this nephron segment in a dose-dependent manner. Stimulation of the enzyme activity was greatest with a maximal dose of PTH and least with VP. With maximal doses, the effects of PTH and CT were not additive; whether or not the effects of maximal doses of VP and PTH or CT were additive was not clear. All three hormones increase cAMP in intact CTAL ina dose-dependent manner. Maximal doses of PTH, CT, VP, and GL resulted in comparable rises in cell cAMP, and there was no additive effect. These data suggest that PTH and CT may stimulate the same adenylate cyclase moieties, whereas VP may stimulate distinct enzyme moieties, and that these three peptide hormones as well as GL definitely act on the same cell group in rat CTAL. Thus, it is possible that these hormones may induce qualitatively similar effects on CTAL functions if such effects are mediated by cAMP.