The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously has been applied to a study on the kinetics of the course of inactivation of yeast alcohol dehydrogenase (YADH) by o-phthaldehyde(OPTA). The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. The inactivation is a monophasic pseudo-first-order reaction with OPTA. The apparent rate constant A is independent of the OPTA concentration, indicating that the inactivation is a noncomplexing inhibition. The marked protective effect of substrates on the inactivation of YADH by OPTA has been observed. This result suggests that the modification of the enzyme by OPTA may occur at the active site.