This study was undertaken to investigate whether chondroitin sulfate (CS) can be efficiently isolated from broiler chicken by-product from mechanical deboning (BMD), which is a mixture of crushed bone, cartilage, skin, adipose tissue and muscle. With BMD as a starting material, it may be possible to maximize CS production from chicken cartilage. CS was liberated from BMD by proteolysis using proteolytic activities from papain, pancreatin, kiwi fruits and flavourzyme. The final product isolated by anion-exchange chromatography and differential precipitation with varying concentrations of ethanol contained CS-peptides with glucuronosyl-N-acetylgalactosamine-4-sulfate as a predominant disaccharide. All the enzyme sources used had sufficient activities to produce CS-peptides with undetectable amount of glucosamine. However, the size of peptide attaching to CS chain and the antigenicity to anti-keratan sulfate (KS) monoclonal antibody varied among the CS-peptides prepared with different proteinases. Papain digestion resulted in the smallest size of peptide with no detectable antigenicity to the anti-KS antibody. In contrast, pancreatin digestion provided CS-peptide having a very weak but positive antigenicity to the anti-KS antibody, which was much lower than that seen with CS-peptide prepared with kiwi fruit proteinase or flavourzyme. Pancreatin is suggested as an economical source of proteinase to liberate CS-peptides from BMD. (C) 2012 Elsevier Ltd. All rights reserved.