DNA probes for trimethoprim-resistant dihydrofolate reductases types IV and V were constructed and characterized. The type IV probe consisted of a 1.7 kb ClaI fragment of recombinant plasmid pUK1148, while the type V probe was a 0.5 kb HincII fragment of plasmid pLK09. Each probe was biotinylated and tested in non-isotopic hybridization experiments with control plasmids carrying genes for known dihydrofolate reductase types. Both probes were highly specific and will therefore be useful in epidemiological studies for monitoring the spread of these unusual plasmid-encoded enzymes.