Because free-insecticide treated net distribution is planned in Benin (West Africa) during the next few years, we investigated the type, frequency and distribution of insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus mosquitoes in four localities selected on the basis of contrasting agricultural practices, use of insecticides and environment. Bioassays with WHO diagnostic test kits were carried out using pyrethroid, carbamate, organophosphate and organochlorine insecticides. An. gambiae mosquitoes were identified to species and to M or S molecular forms using PCR techniques. Molecular and biochemical assays were carried out to identify kdr and Ace.1 mutations in individual mosquitoes and to detect any increase in the activity of enzymes typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transférases). WHO diagnostic tests showed high frequency of resistance in An. gambiae and Cx. quinquefasciatus to permethrin and DDT in three areas. This was consistent with the presence of target site insensitivity due to kdr mutation and to increased metabolism through enzymatic activity. Kdr was expressed in both M and S forms. However, less than 1% of An. gambiae or Cx. quiqnuefasciatus showed the presence of the Ace.1(R) mutation. Carbamate/OP resistance was present at higher frequency in Culex than in An. gambiae. Dieldrin resistance was present in both species at all four localities. A higher frequency of pyrethroid-resistance was found in An. gambiae mosquitoes collected in urban areas compared to those collected in rice growing areas. The expansion of vegetable growing within urban areas probably contributed to selection pressure on mosquitoes. The detection of multiple resistance mechanisms in both An. gambiae and Cx. quinquefasciatus in Benin may represent a threat for the efficacy of ITNs and other forms of vector control such as indoor residual spraying in the future.