A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and spectinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extreme specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA. To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristic of the E. coli chromosome.