The recent discovery that mutation Asn21 --> Ile in the human cationic trypsinogen (Tg) is associated with hereditary pancreatitis has brought into focus the functional role of amino acid 21 in mammalian Tgs. In the present paper, the effect of mutations Thr21 --> Asn and Thr21 --> Ile on the Ca2+ dependence of zymogen activation was investigated, using the autolysis-resistant rat Tg mutant Arg117 --> His. In the absence of Ca2+, rat Tg exhibited low but significant basal autoactivation, which was inhibited by micromolar concentrations of Ca2+ (IC50 2.6 mu M) Interestingly, basal autoactivation was diminished in both mutants, and no further inhibition by micromolar Ca2+ was detectable. Millimolar Ca2+ concentrations markedly and comparably stimulated autoactivation of wild-type and mutant zymogens (ECS, 1.7-2.4 mM). The results indicate that rat Tg is subject to dual regulation by Ca2+, allowing zymogen stabilization in a low-Ca2+ environment and efficient activation in a high-Ca2+ milieu.