The role of steroids in the regulation of colonic sodium transport was examined by infusing steroids into adrenalectomized (ADX) rats and evaluating the short-circuit current (ISC) in vitro. Amiloride-sensitive ISC was induced by aldosterone and corticosterone with half-maximal doses (ED50) of 2 and 260 micrograms X kg-1 X h-1), respectively. Synthetic glucocorticoids such as methylprednisolone (33 mg/kg) and dexamethasone (ED50 = 30 micrograms X kg-1 X h-1) were also effective. Supramaximal doses of aldosterone (7.5 times ED50) for 24 h increased the total ISC (7-fold), the amiloride-sensitive ISC (366-fold), and the conductance (2-fold), as well as the potassium-stimulated phosphatase activity (2-fold) (reported previously). Compared with aldosterone, supramaximal doses of dexamethasone (4 times ED50) produced greater increases in the total ISC (15-fold) and the amiloride-sensitive ISC (674-fold). In contrast to aldosterone, dexamethasone also increased the amiloride-insensitive ISC (3-fold). Glucocorticoid action was not mediated by insulin since the ISC from diabetic ADX rats was increased by dexamethasone to a similar extent (11-fold) as in nondiabetic rats. Estradiol, progesterone, and testosterone did not stimulate the colonic ISC of ADX rats. The ED50 values of corticosterone and aldosterone, measured in terms of amiloride-sensitive sodium transport, produced serum levels that were slightly above those of unstressed, adrenal-intact animals and thus must be considered physiological. It is concluded that at physiological levels both steroids may mediate amiloride-sensitive sodium transport in the rat colon. However, as judged from changes in serum steroid levels, aldosterone is the physiological regulator of elevated sodium absorption in sodium deficiency.