Resistance to temephos, an organophosphorous insecticide (OP), was found to be low (2-fold) in 2 Culex pipiens populations collected in Sayada (mid-eastern Tunisia). This resistance was synergized by an esterase inhibitor (DEF). Two sets of over-produced esterases (A2-B2 and A4-B4), known to be involved in resistance, were identified in almost 50% of the examined insects. In addition, 3% of insects had an insensitive acetylcholinesterase. After selecting larvae of one of the samples (ES) with temephos for 6 generations, a 9-fold increase in resistance was observed, and all mosquitoes were found to carry esterases A2-B2 and an insensitive acetylcholinesterase. These results must be considered in future mosquito control programs, since 2 of the identified genes can lead to high resistance to several organophosphorous insecticides.