Oligopeptidase B is a member of a new serine peptidase family, unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides. An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a variety of conditions. To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E. coli XL1-Blue, and purified essentially in two chromatographic steps. The denatured enzyme failed to refold, which precluded the calculation of free energy of stability, deltaG0. Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat. Denaturation processes were monitored by intrinsic fluorescence, circular dichroism, and activity measurements. A static method, intrinsic fluorescence vs. pH, was indicative of significant changes in the tertiary structure of the enzyme pH < 6 and pH > 8.5. The more sensitive dynamic methods, unfolding rates in urea and inactivation rates at high temperature, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not show significant changes. Inactivation of the enzyme in the acidic pH range correlated with the results obtained with the static rather than with the dynamic method. Acid denaturation at pH 3 was markedly retarded by 1 M NaCl. Against heat inactivation the enzyme was also considerably protected in the presence of salt, and the higher enthalpy and entropy of activation suggested the importance of hydration in the stabilization. The kinetics of unfolding followed single-exponential decay under strongly denaturing conditions (high urea concentration or high temperature), but deviated from the apparently two-state mechanism at low urea concentrations and at slightly acidic pH. The results indicate that under harsher denaturing conditions there is a single rate-limiting step in unfolding, whereas under milder conditions partly unfolded intermediates are populated.