To evaluate the role of endopeptidase 24.11 in metabolism of atrial natriuretic peptide (ANP) in vivo, we examined the effect of thiorphan, an inhibitor of this enzyme, on plasma ANP concentrations and the cardiovascular and renal actions of ANP(99-126). Thiorphan alone produced a modest increase in urinary sodium excretion in anesthetized rats; however, urine flow, arterial pressure, and basal plasma ANP concentrations were unchanged. When administered during an infusion of ANP(99-126) (330 ng/kg/min i.v.), thiorphan increased the plasma concentration of ANP and enhanced the diuretic and natriuretic activity of this hormone. The effects on urine flow and urinary sodium excretion were most pronounced immediately after the inhibitor was administered and later diminished in magnitude. Thiorphan did not alter the depressor activity of exogenous ANP(99-126). These data suggest that endopeptidase 24.11 participates in metabolism of ANP(99-126) and that thiorphan potentiates the renal actions of this hormone by inhibiting its degradation.