Halohydrin dehalogenase from Agrobacterium radiobacter AD1 is a homo-tetrameric protein containing three cysteines per 28 kDa subunit. Under oxidizing conditions the enzyme was found to be susceptible to inactivation which could be prevented by the addition of beta-mercaptoethanol or glycerol. Gel filtration experiments and SDS-PAGE analysis revealed that inactivation coincided with monomerization and intramolecular disulfide bond formation. To identify the cysteine residues involved in the inactivation process, a set of cysteine mutant enzymes was constructed. All the purified mutants (C30A, C153S, C229A and C153S/C229A) showed a similar activity as wild-type enzyme, indicating that no cysteine is directly involved in catalysis. The C153S and C30A mutants displayed a higher stability than wild-type enzyme, whereas mutating Cys229 resulted in decreased enzyme stability. SDS-PAGE analysis showed that in wild-type enzyme Cys30-Cys229 and Cys153-Cys229 disulfide bonds were readily formed while almost no formation of the Cys30-Cys153 disulfide bond could be observed. From this, it was concluded that all three cysteine residues are involved in the enzyme inactivation process. The importance of the improved stability of the C153S and C30A mutant enzymes was demonstrated by performing kinetic resolution experiments with racemic 2-chloro-1-phenylethanol, which resulted in higher enantiomeric excess values of the remaining halohydrin when compared to conversions catalyzed by wild-type enzyme.