pTIM3 is a suicide plasmid vector for the delivery of a transposition defective derivative of Tn5, expressing inducible mercury resistance (HgR) and catechol 2,3-dioxygenase (C23O) activity, to a range of gram-negative microorganisms. pTIM3 was constructed by a four-stage process from pNMM1, a derivative of pSUP5011 containing a modified Tn5where antibiotic-resistance determinants have been replaced by themeroperon of Tn501andtdnC(encoding a C23O). In pTIM3,tdnCis fused tomerD, to bring C23O expression under the control of themeroperon. pTIM3 was introduced intoAcinetobacter calcoaceticus, Pseudomonas putida, Burkholderia cepacia,andAlcaligenessp., and isolates were examined for loss of the vector, which is not stably maintained outside theEnterobacteriaceae,but retention of the Tn5derivative. Transposition results from the action of a vector-encoded Tn5transposase (Tnp) on the ends of IS50Land IS50Rretained in the Tn5construct. Between 10 and 20% of the isolates contained a single copy of the defective transposon in the chromosome. A single-copy isolate of each species was assayed for inducibility of C23O activity using HgCl2as inducer. The increase in C23O activity was linear with increasing HgCl2concentration and ranged between 10- and 20-fold at 20 μg/ml. HgRand C23O+phenotypes were found to be stably maintained in each of the isolates following 40 generations of nonselective growth. A novel aspect of this marker gene system is that isolates can be recovered on nonselective medium and then sprayed with a catechol/HgCl2mixture for rapid colorimetric detection. The system can be applied to the tracking of genetically modified microorganisms in the environment and has the advantage that their detection may be achieved cheaply and without the use of sophisticated equipment.