In a recent survey of trimethoprim resistance, 357 Gram-negative aerobic organisms were isolated from healthy volunteers from rural and urban populations in South Africa. Trimethoprim resistance was transferable in 184 (52%) of the isolates. A further 12 (3%) transferred in the presence of an X+ actor. The transconjugants were probed with intragenic oligonucleotide probes for the type Ia, Ib, IIIa, VIII, V, VI, VII, IX, X and XII dihydrofolate reductase genes. Contrary to all previous data, the most prevalent dihydrofolate reductase gene was the type Ib (30%) followed by the type VIII (23%), V (13%), Ia (6%), VII (3%), and XII (0.5%). None of the strains hybridised to the type IIIa, XI, X and the VI dihydrofolate reductase probes. Plasmid restriction profiles revealed that the high prevalence of the type Ib and VIII dihydrofolate reductase genes resulted from the presence of ubiquitous plasmids. These results highlight the previous problems associated with the distinction of closely related dihydrofolate reductase genes.