Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94-96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as chymotrypsin B1, chymotrypsin B2, chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.