The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp19-22 in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala-replacements of Asp19-22 had minimal or no effect on trypsinogen activation catalyzed by human enteropeptidase. Strikingly, a tetra-Ala19-22 trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, enteropeptidase. In contrast, an intact Asp19-22 motif was critical for autoactivation control. Thus, single-Ala mutations of Asp19, Asp20 and Asp21 resulted in 2-3-fold increased autoactivation, whereas the Asp22->Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala19-21 and tetra-Ala19-22 mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp218, which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp218 in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp19-22 motif per se is not required for enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp218, which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.