Resistance to trimethoprim was surveyed in 2914 Gram-negative bacteria isolated in three hospitals in South Africa. Bacteria were collected from November 1986 to January 1987 and the minimum inhibitory concentration (MIC) of trimethoprim for each isolate was determined. The overall resistance rate (MIC greater than 8 mg/l) was 56.2%, and high-level resistance (MIC greater than 1024 mg/l) occurred in 24.0% of the total. The frequency of resistance in isolates of Enterobacteriaceae was 48.5% (MIC greater than 8 mg/l). Of the organisms isolated from urine specimens, 49.1% were resistant to trimethoprim, 71.8% of these being highly resistant. Investigation of 36 isolates for the presence of the type I and/or type II dihydrofolate reductase genes showed that eight isolates reacted with the type I probe but none with the type II probe.