This study was conducted to identify and characterize antioxidant peptides from the alcalase hydrolysate of the blue-spotted stingray. Purification steps guided by ABTS cation radical (ABTS') scavenging assay and de novo peptide sequencing produced two peptides, WAFAPA (661.3224 Da) and MYPGLA (650.3098 Da). WAFAPA (ECa50 = 12.6 mu M) had stronger antioxidant activity than glutathione (EC50 = 13.7 mu M) and MYPGLA (EC50 = 19.8 mu M). Synergism between WAFAPA and MYPGLA was detected. WAFAPA and MYPGLA surpassed carnosine in their ability to suppress H2O2-induced lipid oxidation. The peptides protected plasmid DNA and proteins from Fenton's reagent-induced oxidative damage. Thermal (25-100 degrees C) and pH 3-11 treatments did not alter antioxidant activity of the peptides. MYPGLA maintained its antioxidant activity after simulated gastrointestinal digestion, whereas WAFAPA showed a partial loss. The two peptides may have potential applications as functional food ingredients or nutraceuticals, whether used singly or in combination.