Transposon Tn4132, encoding the type Ib trimethoprim-resistant, dihydrofolate reductase, was transposed from the clinical plasmid pUK163 to a small recombinant plasmid pZMR12. Restriction endonuclease and partial sequence analysis of Tn4132 revealed a close relationship to Tn7. The nucleotide sequence of the dhfrIb trimethoprim-resistance gene was determined and the gene and its product were found to share significant homology with the dhfrIa, V, VI and VII plasmid-encoded dihydrofolate reductases. Extensive sequence homology (88%) was observed with the type V dihydrofolate reductase at both the nucleotide and amino acid level. Oligonucleotide probes, distinguishing between the dhfrIb and dhfrV genes, were designed. The discriminatory capabilities of these probes in future epidemiological studies will permit a more accurate determination of the dissemination of these two closely related trimethoprim-resistance genes than has previously been possible.