Trypsin from the pyloric ceca of Atlantic bonito (Sarda sarda) was purified and characterized with respect to its purity; molecular weight; sensitivity to temperature, pH, and inhibition; and N-terminal sequence. The purified trypsin had a molecular weight of 29 kDa as per sodium dodecyl sulfate polyacrylamide gel electrophoresis, and optimal activity was observed at pH 9 and 65 degrees C with BAPNA as a substrate. The enzyme was stable to heat treatment up to 50 degrees C and within the pH range of 7-12. It was stabilized by calcium ions, but its activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone, and phenyl methyl sulfonyl fluoride. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration (0-30%). The N-terminal 20 amino acid residues of Atlantic bonito trypsin were determined as IVGGYECQAHSQPWQPVLNS and were homologous with other trypsins.