Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC50 of 7.30 x 10(-7) mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (-20-60 A degrees C), showed > 80% activity across a broad pH range of 4-12 (optimal at pH 4-5) and appeared as a competitive inhibitor of ACE (K (i) of 9.1 x 10(-5) mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC50 of 0.38 A +/- 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC50 of 0.011 A +/- 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K (i) of 1.25 x 10(-6) mg protein/mL).