Among a collection of clinical Escherichia coli isolates, the type I dihydrofolate reductase (DHFR) mediating trimethoprim resistance was generally observed to be chromosomally determined. Only a minority of isolates carried the type I DHFR gene simultaneously on a plasmid. The majority of E. coli isolates studied also hybridized with a probe specific for the transposition gene tnsC of transposon Tn7; and in most of these isolates, Tn7 was found to be inserted into a preferred site in the E. coli chromosome. A minority of isolates that harbored the type I DHFR gene in the chromosome lacked a complete Tn7. Some of these harbored the type I DHFR gene inserted in a structure similar to that containing the gene for streptomycin resistance in Tn21. In the other isolates that were negative for a complete Tn7, the sequences upstream of the type I DHFR gene were demonstrated to be homologous to those flanking the type I DHFR gene in Tn7. This could indicate that the antibiotic resistance region of Tn7 may occur independently of this transposon. In two isolates, no sequences resembling Tn7 or Tn21 were found adjacent to the type I DHFR gene.