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Introduction: The efficacy of Vectobac GR (potency 200 ITU/mg), a new formulation of bacterial larvicide Bacillus thuringiensis var. israelensis Strain AM65-52, was evaluated against Anopheles gambiae and Culex quinquefasciatus in simulated field and natural habitats in Benin., Methods: In simulated field conditions, Vectobac GR formulation was tested at 3 dosages (0.6, 0.9, 1.2 g granules/m² against An. gambiae and 1, 1.5, 2 g granules/m² against Cx. quinquefasciatus) according to manufacturer's product label recommendations. The dosage giving optimum efficacy under simulated field conditions were evaluated in the field. The efficacy of Vectobac GR in terms of emergence inhibition in simulated field conditions and of reduction of larval and pupal densities in rice fields and urban cesspits was measured following WHO guidelines for testing and evaluation of mosquito larvicides., Results: Vectobac GR caused emergence inhibition of ≥80% until 21 [20]-[22] days for An. gambiae at 1.2 g/m² dose and 28 [27-29] days for Cx. quinquefasciatus at 2 g/m² in simulated field habitats. The efficacy of Vectobac GR in natural habitats was for 2 to 3 days against larvae and up to 10 days against pupae., Conclusions: Treatment with Vectobac GR caused complete control of immature mosquito within 2-3 days but did not show prolonged residual action. Larviciding can be an option for malaria and filariasis vector control particularly in managing pyrethroid-resistance in African malaria vectors. Since use of larvicides among several African countries is being emphasized through Economic Community of West Africa States, their epidemiological impact should be carefully investigated.