The genetic structure of two Ralstonia spp., strain JS705 and strain JS745, isolated from the same groundwater aquifer, was characterized with respect to the degradation capacities for toluene and chlorobenzene degradation. Cosmid library construction, cloning, DNA sequencing and mating experiments indicated that the genes for chlorobenzene degradation in strain JS705 were a mosaic of the clc genes, previously described for Pseudomonas sp. strain B13, and a 5 kb fragment identical to strain JS745. The 5 kb fragment identical to both JS705 and JS745 was flanked in JS705 by one complete and one incomplete insertion (IS) element. This suggested involvement of the IS element in mobilizing the genes from JS745 to JS705, although insertional activity of the IS element in its present configuration could not be demonstrated. The complete genetic structure for chlorobenzene degradation in strain JS705 resided on a genomic island very similar to the clc element (Ravatn, R., Studer, S., Springael, D., Zehnder, A.J., van der Meer, J.R. 1998. Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp. strain B13. J Bacteriol 180: 4360-4369). The unique reconstruction of formation of a metabolic pathway through the activity of IS elements and a genomic island in the chlorobenzene-degrading strain JS705 demonstrated how pathway evolution can occur under natural conditions in a few 'steps'.