Collagen, a structural biopolymer of the extracellular matrix, is known to conceal several bioactive peptides which, when excised, can display physiological actions including angiotensin II-converting enzyme (ACE) inhibition. ACE is a key protease controlling the blood pressure (BP) by cleaving dipeptides from an inactive propeptide to produce angiotensin II, a potent BP regulator. Natural inhibitors of ACE, though less potent, have the advantage of being biocompatible and non-toxic. This study was undertaken to identify such cryptic regions from bovine Achilles tendon collagen. Bacterial collagenase was used to hydrolyze collagen and the hydrolysate was subjected to separation through ion-exchange column chromatography. Fractions were subjected to ACE inhibition assays and further purified by gel permeation chromatography. Two biologically active cryptic peptides were obtained displaying potent inhibition abilities; D1 and E2. The peptides were in the mass range of 1.5-3.5 kDa and the inhibition was found to be competitive. Sequence analysis confirmed a relatively higher % occurrence of amino acids A and N in comparison to collagen and a hydrophobic C-terminal with P as the terminus. Both peptides were found to retain 80% of activity, even after digestive enzyme treatment. IC 50 values revealed D1 to be the most potent inhibitor. Docking studies revealed that both peptides were using the C-terminal to interact with ACE-binding site. A comparison with other peptides displaying competitive inhibition hinted at the presence of a unique sequence GX′Y′ where X′ is often P, L, I or A and Y′ often P as the probable C-terminal for effective inhibition. © 2012 Elsevier Ltd.