The inhibitory activity of angiotensin I-converting enzyme (ACE) was extracted with 80% ethanol from the leaves of Ashitaba (Angelica keiskei). The present ACE inhibitor was fractionated and separated with various chromatographies. The antihypertensive effects of the sample (G fraction) from Ashitaba on spontaneously hypertensive rats (SHR) were observed by long-term administration for 10 wk. Another sample (S fraction) from Ashitaba also had antihypertensive effects after a single intravenous administration to SHR. The sample was further purified by using several chromatographies. The ACE inhibitor fraction was characterized as follows: no significant absorbance, a zwitterion, a water-soluble substance and a positive ninhydrin reaction. According to a mass spectrum analysis, the molecular weight of the ACE inhibitor was determined to be 303 and Na-salt ions of carboxyl groups were detected. The ACE inhibitor from Ashitaba contained in the anti-hypertensive fraction was speculated to be very similar to authentic nicotianamine based on a comparative study of inhibitory activity, mass spectrum analysis and thin-layer chromatographies.