A clinical strain of Escherichia coli isolated in Nottinghamshire in 1980 was shown to harbour the type IIIa trimethoprim-resistant dihydrofolate reductase gene, previously identified on only one occasion, in New Zealand in 1979. The gene was identified by hybridisation with an 855-bp type III gene probe and its classification as a type IIIa dihydrofolate reductase was confirmed by detailed biochemical analysis of the enzyme product. The dihydrofolate reductase was identical in size and isoelectric point with the original type IIIa enzyme and shared similar inhibitory and kinetic profiles. The trimethoprim resistance gene was subsequently cloned and the type IIIa dihydrofolate reductase gene was localised to a 700-bp EcoRI-PstI fragment. This smaller fragment may prove to be a more specific DNA probe for the future identification of type IIIa dihydrofolate reductase genes.