The XylR regulatory protein is a transcriptional activator from the TOL plasmid of Pseudomonas putida mt-2 that is involved in the toluene and benzene degradation pathway. Here we describe the construction and laboratory characterization of recombinant biosensors (pGLPX plasmids) based on XylR and its cognate promoter (Pu). In the pGLPX plasmid, the reporter luc gene is under the control of the Pu promoter. We evaluated the ability of two distinct nucleotide sequences to function as SD elements and improve sensitivity of bioreporting. We also evaluated the effect of introducing the T(2)rrn beta terminator on the specificity of the construct. E. coli transformed with pGLPX plasmids were used to sense toluene and its derivatives. The pattern of induction was different for each derivative. In general, more luciferase activity was induced by toluene and benzene than by TNT and DNT at most tested concentrations. The bioluminescence response of the reporter strains to the nitrotoluenes was significantly stronger at lower concentrations (a parts per thousand yen50 mu mol) than at higher concentrations. Our results show that the SD sequence (taaggagg) is crucially important for biosensor sensitivity. The presence of the T(2)rrn beta terminator in the bioreporter plasmid prevents nonspecific responses and also reduces biosensor sensitivity upon exposure to inducers. These data suggest that pGLPX strains can be used as whole-cell biosensors to detect toluene and related compounds. Further investigation will be required to optimize the application of pGLPX biosensors.