Activities of proteolytic enzymes, represented by azocasein digestion (total protease), trypsin, and chymotrypsin from the gastric fluid of the marine crab, Cancer pagurus, were evaluated for operational parameters like pH-optimum, thermal stability, and effectors such as metal ions and organic solvents. Total protease activity was 98.5±4.3 ΔE366 min-1 ml-1. Trypsin and chymotrypsin activities amounted to 14.7±1.5 U ml-1 and 197±59 U ml-1, respectively. The maximum activity of total protease appeared at pH 5-7, that of trypsin at pH 7-9, and that of chymotrypsin at pH 6.5-7. Trypsin and chymotrypsin activities were slightly activated by Ca2+, but were drastically reduced by Zn2+. In the presence of 2-propanol, trypsin activity was enhanced 8-fold at 30°C. Highest amplification of activity of up to 30-fold, however, appeared below 12°C. The effects of ethanol and acetone were less distinct. These caused a 2.5-fold increase of activity. Methanol was the least activating solvent. Activities of all enzymes remained stable for up to 127 days at 5°C, keeping up to 70% of the initial activity. When stored at 25°C for 120 days, total protease decreased to about 40% and trypsin and chymotrypsin activities kept more than 60% of initial activity. Zymograms showed five major and several minor proteolytic bands with apparent molecular weight of 20-45 kDa. After long-term storage at room temperature, the major bands remained active, while some bands with minor activity disappeared. This proteolytic enzyme preparation from the gastric fluid of crab seems suitable for biotechnological application at neutral pH and in the presence of organic solvents. © 2004 Elsevier B.V. All rights reserved.