The aim of this study was to facilitate development of natural antioxidants from defatted peanut kernels. Protein hydrolysates obtained from defatted peanut kernels with esperase treatment for 2 h exhibited higher antioxidative activity toward linolenic acid peroxidation than other proteases (including neutrase, pepsin, protease A and protease N). The esperase hydrolysate of peanut protein (EHPP) was further separated with 3 and 5 kDa molecular cut-off membranes to determine the influence of molecular weight. EHPP with molecular weight 3 similar to 5 kDa showed higher relative antioxidative activity, DPPH radical scavenging activity and metal chelating activity than that with molecular weight lower than 3 kDa or higher than 5 kDa. The fraction was further purified by ion exchange chromatography and high-performance liquid chromatography; the final peanut peptides exhibited higher relative antioxidative activity (27.5) than ascorbic acid (9.5). This study reported for the first time that protein hydrolysates from defatted peanut kernels possess antioxidative activity. (C) 2009 Elsevier Ltd. All rights reserved.