The effect of the addition of rennet from different origin (animal, plant and microbial) on the release of potential antioxidant peptides was evaluated in hard cow milk cheese. After cheese making, chemical parameters, colour, TBARs and riboflavin content were determined in the final product. Antioxidant activity of the water soluble extracts from cheese was evaluated by the DPPH radical scavenging and metal chelating activity assays. Separation of peptide fractions (<3 kDa) was performed by RP-HPLC and fractions with potential antioxidant activity were analyzed by LC-MS/MS for further identification of peptides. Results suggested that colour and riboflavin content were affected by the type of rennet used (P <.05). On the other hand, extracts of cheese with microbial rennet showed the highest antioxidant activity (P <.05). Only nine fractions isolated from the extracts showed either DPPH radical scavenging or metal chelating activities. Peptides EIVPN and DKIHPF, previously reported in other studies by showing ACE-inhibitory activity, were identified in these fractions and revealed high metal chelating activity. Moreover, a new peptide, with also high metal chelating activity, has been identified (VAPFPQ). In conclusion, it would be worthwhile to synthesis these peptides and confirm their antioxidant and metal chelating activities.