Resistance in Anopheles gambiae to members of all 4 major classes (pyrethroids, carbamates, organochlorines, and organophosphates) of public health insecticides limits effective control of malaria transmission in Africa. Increase in expression of detoxifying enzymes has been associated with insecticide resistance, but their direct functional validation in An. gambiae is still lacking. Here, we perform transgenic analysis using the GAL4/UAS system to examine insecticide resistance phenotypes conferred by increased expression of the 3 genes— Cyp6m2 , Cyp6p3 , and Gste2 —most often found up-regulated in resistant An. gambiae . We report evidence in An. gambiae that organophosphate and organochlorine resistance is conferred by overexpression of GSTE2 in a broad tissue profile. Pyrethroid and carbamate resistance is bestowed by similar Cyp6p3 overexpression, and Cyp6m2 confers only pyrethroid resistance when overexpressed in the same tissues. Conversely, such Cyp6m2 overexpression increases susceptibility to the organophosphate malathion, presumably due to conversion to the more toxic metabolite, malaoxon. No resistant phenotypes are conferred when either Cyp6 gene overexpression is restricted to the midgut or oenocytes, indicating that neither tissue is involved in insecticide resistance mediated by the candidate P450s examined. Validation of genes conferring resistance provides markers to guide control strategies, and the observed negative cross-resistance due to Cyp6m2 gives credence to proposed dual-insecticide strategies to overcome pyrethroid resistance. These transgenic An. gambiae -resistant lines are being used to test the “resistance-breaking” efficacy of active compounds early in their development.