Long-lasting insecticidal nets (LLINs) may eliminate the need for retreatment of mosquito nets used for the control of malaria and other vector-borne diseases. The efficacy of LLINs after repeated washing under laboratory conditions has been used to predict long-lasting efficacy under field conditions. We evaluated under laboratory conditions the wash resistance of two LLINs (PermaNet 1.0, Vestergaard-Frandsen, Denmark; Olyset, Sumitomo Chemical Co., Japan), two candidate LLINs (Dawa, Siamdutch Mosquito Netting Co., Thailand; Insector, Athanor, France) and a net treated with a process designed to increase its wash resistance and compared them with conventionally treated nets (deltamethrin, 25 mg/m(2)). Nets of all six types were washed using a standard protocol and tested weekly using WHO cone bioassays with Anopheles gambiae (Kisumu strain). The PermaNet 1.0 was the most wash resistant with >50% mosquito mortality in WHO cone bioassays after as many as 20 washes. The Dawa net also retained some activity after repeated washing but exhibited wide variation in insecticide retention and biological activity. The remaining nets lost >90% of their biological activity after six washes as measured by 24-h mortality of A. gambiae in WHO cone tests. After 20 washes, all nets lost >50% of their initial insecticide concentrations except for the Olyset net. After 20 washes, nets were heated for 4 h at 60 degrees C to determine whether biological activity could be restored by heat-assisted regeneration. Only the Olyset net was regenerated by heating, with average mosquito mortality and knockdown in WHO cone tests rising to >90% after heating for 4 h at 60 degrees C. However, regeneration of the biological activity of Olyset nets that had been washed three times did not occur at 30 degrees C or 35 degrees C after 12 weeks. The wash resistance of these LLINs corresponded well to their retention of biological activity observed in a field trial, suggesting that wash resistance may be a good predictor of the longevity of insecticidal activity of LLINs under field conditions.