Jumbo squid specimens were captured, dissected, and the hepatopancreas was freeze dried for the extraction of proteolytic enzymes. An autolysis experiment conducted at 25C showed two peaks with maximum proteolysis at pH 3 and 5. The proteinase activity of the extract was measured using azocasein, BANA, Z-PAAFC and Z-AAAFC substrates. Activity of the extract with azocasein (pH 5) had a maximum at 55C and increased threefold with the inclusion of cysteine or DTT. The proteinase activity remained at least at 60% of the original after 45 h at 4C in the pH range of 3-8. Activity was inhibited 70-85% when extracts were treated with cysteine proteinase specific inhibitors. The proteinases extracted from jumbo squid hepatopancreas are mainly of the cysteine type and have significant activity towards a cathepsin L specific substrate. The understanding of proteinases from this tissue could have implications for quality control of jumbo squid food products. © Copyright 2005, Blackwell Publishing.