The malaria control programme of KwaZulu-Natal Province, South Africa, includes Mamfene and Mlambo communities. Western-type houses there are currently sprayed with deltamethrin, whereas traditional houses are sprayed with DDT for malaria control. In 2002, mosquitoes of the Anopheles gambiae complex (Diptera: Culicidae) were collected from DDT-sprayed houses, by window exit traps, and from man-baited nets outdoors. Larval collections were also carried out at Mzinweni Pan near Mlambo. Species of the An. gambiae complex were identified by rDNA polymerase chain reaction assay. The majority of samples collected by window trap and baited nets were identified as the malaria vector An. arabiensis Patton, with a few An. merus Dönitz and An. quadriannulatus (Theobald). The larval collections were predominantly An. quadriannulatus with a small number of An. arabiensis. Standard WHO insecticide susceptibility tests using 4% DDT and 0.05% deltamethrin were performed on both wild-caught females and laboratory-reared progeny from wild-caught females. Wild-caught An. arabiensis samples from window traps gave 63% and 100% mortality 24-h post-exposure to DDT or deltamethrin, respectively. Wild-caught An. arabiensis samples from man-baited net traps gave 81% mortality 24-h post-exposure to DDT. The F1 progeny from 22 An. arabiensis females showed average mortality of 86.5% 24-h post-exposure to DDT. Less than 80% mortality was recorded from five of these families. Biochemical analyses of samples from each of the families revealed comparatively high levels of glutathione-S-transferases and non-specific esterases in some families, but without significant correlation to bioassay results. Wild-caught An. quadriannulatus larvae were reared through to adults and assayed on 4% DDT, giving 47% (n = 36) mortality 24-h post-exposure. Finding DDT resistance in the vector An. arabiensis, close to the area where we previously reported pyrethroid-resistance in the vector An. funestus Giles, indicates an urgent need to develop a strategy of insecticide resistance management for the malaria control programmes of southern Africa.