The fluorescence of pepsin-bound 2-p-toluidinylnaphthalene-6-sulfonate (TNS) decreases upon the binding of Streptomyces pepsin inhibitor (SPI) with the enzyme. Equilibrium dialysis experiments showed this decrease to arise from the release of TNS from pepsin due to the binding of SPI in a molar ratio of 1:1. The results of stopped-flow kinetic study on the concentration dependence of the apparent rate constant for the binding reaction between pepsin and SPI using the fluorescence decrease of TNS as a probe were consistent with the interpretation that the binding reaction consists of at least two steps, including a fast bimolecular step followed by a slow unimolecular step. It was found that a fluorescence decrease occurred in both steps. The rate-pH profile suggested the participation of some ionizable group(s) (possibly a carboxyl group) in the binding.