A speciation procedure for chromium(III) and chromium(VI) in the environment al samples has been established in the presented work. The procedure presented based on quantitative biosorption of chromium(III) on Bacillus sphaericus loaded Diaion SP-850 at pH 5. The Cr(VI) recoveries at pH 5 were below 5% on the biosorbent. After reduction of Cr(VI) by concentrated H2SO4 and ethanol, the system was applied to the total chromium. Cr(VI) was calculated as the difference between the total chromium content and the Cr(Ill) content. Optimal analytical conditions including pH, amounts of biosorbent, etc. for Cr(Ill) recoveries were investigated. The influences of the some alkaline and earth alkaline ions and some transition metals on the recoveries were also investigated. The capacity of biosorbent for chromium(III) was 6.95 mg g(-1). The detection limit (3 sigma) of the reagent blank for chromium(III) was 0.50 mu g L-1. The procedure was successfully applied to the speciation of chromium(III) and chromium(VI) in natural water samples (R.S.D. lower than 5%, recoveries greater than 95%). (C) 2006 Elsevier B.V. All rights reserved.