Antibodies were prepared in rabbits against trypsins obtained from four different species-namely bovine, porcine, spiny Pacific dogfish, and starfish (Evasterias trochelii). Each of the antisera, or the immunoglobulin G fraction thereof, was tested for its capacity to react with each of the four enzymes. The immunological reaction was assessed by three different techniques-precipitin reaction, antigen binding capacity, and inhibitory effect of the antibodies on the proteolytic activity of the enzymes. In each case, the homologous enzyme gave the strongest reaction with its antiserum but the heterologous enzymes were also capable of reacting to an appreciable extent. The cross-precipitation was the least sensitive method, and gave relatively low values, whereas cross-inhibition and cross-binding of radioactively labeled antigens indicated high extent of cross-reaction between the various trypsins. The relative capacity of interaction of the four enzymes with the four antibodies could be related to the order in which these enzymes developed during evolution. Thus the "order" of similarity was bovine > porcine > dogfish > starfish.