A survey was conducted in South Africa to determine the incidence of resistance in aerobic Gram-negative commensal faecal flora. Faecal specimens from 272 out of 362 (75%) healthy volunteers carried trimethoprim-resistant strains, from which 357 trimethoprim-resistant organisms were isolated. Trimethoprim resistance was transferable by conjugation in 55% of the isolates. The majority of the isolates were resistant to other antimicrobial agents including ampicillin 71.4% and tetracycline 88%. Most of these resistance phenotypes co-transferred with trimethoprim resistance. Analysis of 148 plamids revealed 79 different restriction profiles which indicated that there is a large gene pool of trimethoprim-resistant organisms in the faecal flora. High-level resistance to trimethoprim (MIC ≥ 1024mg/l) occurred in 98.6% of the isolates suggesting that resistance in these isolates was mediated by the production of additional trimethoprim resistant dihydrofolate reductase (DHFR) genes. To determine the epidemiology of these genes, oligonucleotide probes were designed from the nucleotide sequence of a heterogeneous region which occurs within all trimethoprim resistant DHFR genes. Hybridisation experiments revealed that contrary to all previous data, the most prevalent DHFR of the transferable genes which hybridised was the type Ib (30%), followed by the type VIII (23%), V (13%), Ia (6%), VII (3%) and XII (0.5%). On the other hand the type VII, (38%) was the most prevalent dihydrofolate reductase gene in the 161 (45%) isolates which did not transfer their resistance factors, followed by type Ia (25%), type Ib (12%), type V (2%) and type VIII (0.5%).