Although the bacterial degradation of chloral hydrate (CH) has been recognized for several decades, its degradation pathway by assimilation has not been demonstrated. In this paper, we report the isolation of the LF54 bacterial strain, which utilizes CH as its sole carbon and energy source. LF54 converted CH into trichloroethanol (TCAol), which was dehalogenated to dichloroethanol (DCAol), and CO(2) was detected as the end product. Another strain that we isolated, RS20, co-metabolized CH into TCAol. Our 16S rRNA gene sequencing and taxonomic analyses revealed that the LF54 and RS20 strains belong to the Pseudomonas and Arthrobacter genera, respectively. When the two strains were inoculated into soil microcosms, both degraded 0.3 mM CH to undetectable levels (<0.01 mM) within 5 days. These results suggest that LF54 and RS20 could be used in the bioremediation of CH-contaminated environments. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.