An amperometric bacterial sensor with current response to Fe(2+) and S(2)O(3)(2-) ions has been designed by immobilizing an acidophilic biomass of Acidithiobacillus ferrooxidans on a multi disk flat-front oxygen probe. The bacterial layer was located between the oxygen probe and a membrane of cellulose. A filtration technique was used to yield the bacterial membranes having reproducible activity. The decrease of O(2) flow across the bacterial layer is proportional to the concentration of the dosed species. The dynamic range appeared to be linear for the Fe(2+) ions up to 2.5 mmol L(-1) with a detection limit of 9 x 10(-7) mol L(-1) and a sensitivity of 0.25 A L mol(-1). The response of the biosensor is 84 s for a determination of 2 x 10(-4) mol L(-1) Fe(2+). Optimizing the Fe(2+) determination by A. ferrooxidans sensor was carried out owing to Design of Experiments (DOE) methodology and empirical modelling. The optimal response was thus obtained for a pH of 3.4, at 35 degrees C under 290 rpm solution stirring. S(2)O(3)(2-) concentration was determined at pH 4.7, so avoiding its decomposition. The concentration range was linear up to 0.6 mmol L(-1). Sensitivity was 0.20 A L mol(-1) with a response time of 207 s for a 2 x 10(-4) mol L(-1) S(2)O(3)(2-) concentration.