The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.