Endogenous ouabain-like compounds are synthesized in and released from the adrenal gland. Although endogenous ouabain (EO) has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal has not been elucidated. To address this issue we studied the effects of reduction in plasma EO resulting from anti-ouabain antibody administration. Normal rats were treated for 28 days with anti-ouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannule by osmotic pumps and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the anti-ouabain antibody-treated rats. Moreover, on the second day of treatment there was a significant transient reduction in natriuresis in the anti-ouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-BSA. Furthermore, following 28 days of treatment the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from anti-ouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.