The dhlA gene of Xanthobacter autotrophicus encodes dehalogenase that hydrolyzes dihaloalkanes such as 1,2-dichloroethane (DCE) into cytotoxic halogenated alcohol and an inorganic halide. As plants do not contain dehalogenase activity, they grow normally in the presence of DCE. We tested the transgenic expression of the bacterial dhlA gene in rice as a conditional negative selection marker. We developed 24 transgenic callus lines containing dhlA gene driven by rice actin-1 promoter, verified the expression of dhlA by Northern blot analysis, and subjected these transgenic lines to DCE treatment. We found that, while untransformed callus (Nipponbare) was unaffected by the DCE treatment, most of the transformed lines displayed symptoms of toxicity, indicating that dhlA is an effective conditional negative selection marker gene for rice in vitro cultures.