Strong denaturants can be used to distinguish between heat-induced changes in the primary structure of the enzyme molecule and heat-induced changes in higher orders of structure. In this paper; we report on an attempt to use urea in studying the mechanism of thermal inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F. Addition of urea at >2 M (without heating) resulted in inactivation which was, however; reversible. Diluting to concentrations <2 M urea completely restored proteolytic activity. The rate of inactivation at 100 degrees C of the proteinase was increased when 6 M urea was present during heat treatment. Also at lower urea concentrations, the inactivation rate at 100 degrees C was increased. Addition of 6 M urea to the enzyme solution after heat treatment also increased the extent of inactivation while low urea concentrations (<1 M) did not. It was concluded that cyanate formed from urea at high temperature was the cause of increased inactivation since addition of cyanate could increase the inactivation rate while a treatment to remove cyanate from a heated urea solution could prevent increased inactivation The use of urea does not appear to be suitable for the elucidation of the mechanism of thermal inactivation of the extracellular proteinase from P. fluorescens 22F, but might be applicable to other enzymes when treated (cyanate fi ee) urea is used after heat treatment; however, use of urea (even if cyanate free) during heat treatment is not possible because cyanate is induced by the very heat treatment.