Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 has been shown to degrade all chlorinated ethenes individually and as mixtures. Here, DNA shuffling of the alpha hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues 1100, Q141, T201, F205, and E214 were used to enhance the degradation of chlorinated aliphatics. The ToMO mutants were identified using a chloride ion screen and then were further examined by gas chromatography. Escherichia coli TG1/pBS(Kan)ToMO expressing TouA saturation mutagenesis variant 1100Q was identified that has 2.8-fold better trichloroethylene (TCE) degradation activity (apparent V-max of 1.77 nmol min(-1) mg(-1) protein(-1) vs 0.63 nmol min(-1) mg(-1) protein(-1)). Another variant, E214G/D312N/M399V, has 2.5-fold better cis-1,2-dichloroethylene (cis-DCE) depradation activity (apparent V-max of 8.4 nmol min(-1) mg(-1) protein(-1) vs 3.3 nmol min(-1) mg(-1) protein). Additionally, the bydroxylation regiospecificity of o-xylene and naphthalene were altered significantly for ToMO variants A107T/ E214A, T201G, and T201 S. Variant T201 S produced 2.0-fold more 2,3-dimethylphenol (2,3-DMP) from o-xylene than the wild-type ToMO, whereas variant A107T/E214A bad 6.0-fold altered regiospecificity for 2,3-DMP formation. Variant A107T/E214A also produced 3.0-fold more 2-naphthol from naphthalene than the wild-type ToMO, whereas the regiospecificity of variant T20 I S was altered to synthesize 3.0-fold less 2-naphthol, so that it made almost exclusively 1-naphthol (96%). Variant T201G was more regiospecific than variants A107T/E214A and T201S and produced 100% 3,4-DMP from o-xylene and > 99% 1-naphthol from naphthalene. Hence, ToMO activity was enhanced for the degradation of TCE and cis-DCE and for the regiospecific hydroxylation of o-xylene and naphthalene through DNA shuffling and saturation mutagenesis.