Angiotensin I-converting enzyme (ACE) inhibitors have a potent antihypertensive effect in both humans and animals. Various peptides derived from food proteins were found to have ACE inhibitory activity. For determining ACE inhibitory activity, protein hydrolysates were prepared from tuna broth (contain 4% water-soluble protein) using various commercial proteases. Orientase hydrolysate (OAH) exhibited the most potent inhibitory activity on ACE. The best hydrolysis conditions with respect to inhibition of ACE were 3hr of incubation at 50˚C, pH 6.5, with enzyme (1.0% orientase) to substrate (tuna broth) of 1/25 (v/v). Preparation under these conditions yielded activity (IC50 12.52 ± 0.02 mg/mL). Fractionation of the orientase hydrolysate on a Sephadex G-25 gel filtration chromatograph resulted in the production of more active inhibitors. The active fractions were peptides of molecular weights < 1,000 Da. The most active fraction had an IC50 value of 0.21 ± 0.01 mg/mL. This fraction was rich in basic amino acids and aromatic amino acids [peptides with molecular mass (< 565 Da)].