Chromium-resistant bacteria (CRB) isolated from soils can be used to reduce toxic Cr(VI) from contaminated environments. This study assessed in vitro reduction of hexavalent Cr using a cell-free extract (CFE) of CRB isolated from soil contaminated with dichromate. One isolate, ES 29, that substantially reduced Cr(VI) was identified as a Bacillus species by 16S rRNA gene-sequence homology. The isolate reduced Cr(VI) under aerobic conditions, using NADH as an electron donor and produced a soluble Cr(VI)-reducing enzyme stimulated by copper (Cu2+). The CFE of the bacterial isolate reduced 50% of Cr(VI) in 6 h. The Cr(VI)-reduction activity of the CFE had a Km of 7.09 microM and a Vmax of 0.171 micromol min(-1) mg(-1) protein. Mercury inhibited the enzyme, but not competitively, with a Vmax of 0.143 micromol min(-1) mg(-1) protein, a Km of 7.07 microM and a Ki of 1.58 microM. This study characterizes the enzymatic reduction of Cr(VI) by Bacillus sp. ES 29 which can be used for the bioremediation of chromate.