1. Inhibition of neutral endopeptidase (NEP), the degradative enzyme for atrial natriuretic peptide, was studied in vitro and in vivo using a previously characterized NEP inhibitor radioligand, 125 I-labelled RB104. 2. SCH 42354, the active di-acid of the ethylester prodrug, SCH 42495, caused a concentration-dependent displacement of 125 I-labelled RB104 from rat renal NEP. The concentration of SCH 42354 that displaced 50% of radioligand bound to the enzyme NEP (IC 50 ) was 3.3 ± 0.1 nmol/1 (mean ± SEM). Enalaprilat, an angiotensin converting enzyme inhibitor, did not displace 125 I-labelled RB104 in concentrations up to 10 μmol/1. 3. In adult normotensive Sprague—Dawley rats, oral SCH 42495 (3–300 mg/kg) caused significant inhibition of renal NEP ( P < 0.001). SCH 42495 had no effect on renal or plasma angiotensin converting enzyme activity, but high-dose SCH 42495 (300 mg/kg) caused a significant increase in plasma renin activity ( P < 0.01). 4. In a time course study, oral SCH 42495 (30 mg/kg) caused rapid (within 30 min) and significant inhibition of renal NEP for up to 48 h ( P < 0.001). No changes in plasma atrial natriuretic peptide or plasma angiotensin converting enzyme activity were seen. 5. These data provide evidence that short-term administration of the NEP inhibitor SCH 42495 results in inhibition of renal NEP and does not inhibit the circulating or the tissue renin—angiotensin system. The NEP inhibitor radioligand 125 I-labelled RB104, is a useful tool to study tissue NEP inhibition after administration of NEP inhibitors.