Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentrations (10-20 microM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15 nM). The data indicate that the inhibition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formation of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, respectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule.