The Gram-positive bacterium, Bacillus stearothermophilus, was used as a model organism to identify the non-selective toxic effects of the currently used insecticide methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate). A significant decrease of the yield of bacterial cultures and a premature appearance of ultrastructural abnormalities in cells cultured in the presence of the insecticide were taken as indicators of cytotoxicity. A putative correlation of this cytotoxicity with methoprene-induced perturbations on membrane lipid organization was investigated, using differential scanning calorimetry and the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH-PA). The membrane physical effects depended on the lipid bilayer composition and packing. The most striking effect was a progressive broadening and shifting to lower temperatures, with increasing methoprene concentrations, of the main transition phase of the dimyristoyl- or dipalmitoylphosphatidylcholine bilayers and of the lateral phase separation of liposomes reconstituted with the lipid extracts of B. stearothermophilus.