Environmental contamination by hexavalent chromium, Cr(VI), presents a serious public health problem. This study assessed the reduction of Cr(VI) by intact cells and a cell-free extract (CFE) of an actinomycete, Arthrobacter crystallopoietes (strain ES 32), isolated from soil contaminated with dichromate. Both intact cells and CFE of A. crystallopoietes, displayed substantial reduction of Cr(VI). Intact cells reduced about 90% of the Cr(VI) added within 12 h and Cr(VI) was almost completely reduced after 24 h. The KM and Vmax of Cr(VI) bioreduction by intact cells were 2.61 microM and 0.0142 micromol/min/mg protein, respectively. Cell-free chromate reductase of the A. crystallopoietes (ES 32) reduced hexavalent chromium at a KM of 1.78 microM and a Vmax of 0.096 micromol/min/mg protein. The rate constant (k) of chromate reduction was inversely related to Cr(VI) concentration and the half-life (t1/2) of Cr(VI) reduction increased with increasing concentration. A. crystallopoietes produced a periplasmic chromate reductase that was stimulated by NADH. Results indicate that A. crystallopoietes ES 32 can be used to detoxify Cr(VI) in polluted sites, particularly in stressed environments.