The chromate reductase activity of cytochrome c3 (Cyt c3, Mr 13 000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO42−) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c3 by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l−1 (3.85–132 μM) with a sensitivity of 35 nA mg−1 l (1.82 nA μM−1). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c3 (372 ng of protein) was needed for this biosensor.