OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217±Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO 2 )-NH 2 (where Abz is o-aminobenzoyl and Tyr(NO 2 ) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity K m of 0.4 mm for the substrate and a turnover number k cat of 40 s 21. The K m and k cat for the double variant were 1.1 mm and 1.6 s 21 , respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pK a values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of b-strand conformation, and on that basis a b-barrel topology model is proposed.