Trypsinogen and chymotrypsinogen hydrolyze p-nitrophenyl esters of several peptides and tert-butyloxycarbonyl amino acids. The best substrate found for chymotrypsinogen was Boc-Ala-ONp and for trypsinogen Z-Gly-Hyp-Gly-ONp. Comparison of the kinetic parameters indicates that in the zymogens the catalytic site is distorted and reduced in effectiveness by about two orders of magnitude, in addition to a 10 000-fold decrease in catalysis due to a distortion of the primary substrate binding site (Kerr, M.A., Walsh, K.A., & Neurath, H. (1976) Biochemistry 15, 5566). Using Boc-Ala-ONp as substrate and certain aldehydes and borates as inhibitors, the zymogens were tested for the integrity of the "oxyanion hole", but these results were largely inconclusive. Probes for the secondary binding sites indicated their presence in trypsinogen and their absence in chymotrypsinogen.