Progress curve analysis is applied to the estimation of inactivation rate constants. The heat inactivation of inorganic pyrophosphatase from baker's yeast was studied assuming a simple one-substrate mechanism containing two irreversible inactivation steps for the free enzyme and the enzyme-substrate complex, respectively. Four kinetic parameters including Km simultaneously may be detected from one progress curve. The temperature dependences derived from the estimated rate constants for several inactivating temperatures agree well with that obtained from preincubation measurements.