Formation and dissociation of the benzamidine:beta-trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine:beta-trypsin complex minus the free proteinase) is 1.75 mM-1 cm-1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the beta-trypsin:BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 +/- 0.5 degree C. Under all the experimental conditions the beta-trypsin:BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine:beta-trypsin:BPTI adduct (7). The difference between the kinetic processes of beta-trypsin:BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S1 subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.