A biosensor was developed for the detection of tributyltin (TBT), using a bioluminescent recombinant Escherichia coli::luxAB strain. Dedicated devices allowed the on-line measurement of bioluminescence, pH and dissolved oxygen values and the feed-back regulation of temperature. Bacterial physiology was monitored by the measurement of the cellular density, respiratory activity and the intracellular level of ATP, glucose and acetate levels. Our results showed that a synthetic glucose medium gave a better TBT detection limit than LB medium (respectively 0.02 muM and 1.5 muM TBT). High growth and dilution rates (D=0.9 h(-1)) allowed maximum light emission from the bacterium. Moreover, simple atmospheric air bubbling was sufficient to provide oxygen for growth and the bioluminescence reaction. Real-time monitoring of bioluminescence after TBT induction occurred with continuous addition of decanal up to 300 muM, which was not toxic throughout a 7-day experiment. The design of our biosensor and the optimization of the main parameters that influence microbial activity led to the capacity for the detection of TBT.