Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S-12, S-32, and S-150). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S-150) concentration equivalent to 4.56 mg protein/mL, 300 mu M Cr(VI) and pH 6.0 after 30 min incubation at 30 degrees C. The enzyme was heat labile; 80% of its activity was lost when exposed at 70 degrees C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a V-max of 1.432 mu mol Cr(VI)/mg protein/min and K-m of 158.12 mu M Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity over control was recorded with 1.0 mu M NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 mu M Co(II), which retained 93% activity over control.