Low molecular weight esters are volatile insecticides that possess fumigant-like activity, and which have potential applications in integrated pest management. Here, we report on investigations into the mode of action and metabolism of formate esters by insecticide susceptible and metabolically-resistant Drosophila. In bioassays, the formate esters and their hydrolysis product formic acid generally showed higher levels of toxicity to the susceptible Canton-S strain than to the metabolically-resistant Hikone-R strain. Based on a colorimetric assay using fly homogenates, the rate of hydrolysis of formate esters was higher in Canton-S than Hikone-R. The uptake of formate esters by flies after fumigation was high, and most of the absorbed esters were converted to formic acid in vivo. In agreement with colorimetric assays, higher quantities of formic acid were observed in Canton-S than Hikone-R after fumigation. Cytochrome C oxidase inhibition assays using the standard insecticides sodium cyanide and hydramethylnon showed potent inhibition of Drosophila mitochondrial activity. However, the formate esters as well as other types of volatile esters (acetate, propionate, butyrate and valerate) were much less effective at cytochrome C oxidase inhibition. These results suggest that volatile low molecular weight esters elicit their insecticidal activity at target sites other than cytochrome C oxidase.