Tn5Map, a Tn5 derivative containing the 18 bp I-SceI site, was delivered from a RP4-mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-SceI sites in their genomes. Digestion of the DNA from Tn5Map-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion. These digests also gave direct and independent proof for the single circular chromosome of E. coli, and for the presence of two circular chromosomes in B. melitensis and of a circular and a linear chromosome in A. tumefaciens C58 (which also contains two large circular plasmids). This rapid and versatile technique is potentially applicable to the study of the genomic organization in all Gram-negative bacteria which support Tn5 transposition. Moreover, linearization of circular replicons could be the first step for a rapid method of physical mapping.