Juvenile hormone esterases (JHEs) from six insects belonging to three orders (Lepidoptera, Coleoptera, and Diptera) were compared in terms of their deduced amino acid sequence and biochemical properties. The four lepidopteran JHEs showed from 52% to 59% identity to each other and about 30% identity to the coleopteran and dipteran JHEs. The JHE of Manduca sexta was remarkably resistant to the addition of organic co-solvents and detergent; in some cases, it demonstrated significant activation of activity. Trifluoromethylketone (TFK) inhibitors with chain lengths of 8, 10 or 12 carbons were highly effective against both lepidopteran and coleopteran JHEs. The coleopteran JHE remained sensitive to TFK inhibitors with a chain length of 6 carbons, whereas the lepidopteran JHEs were significantly less sensitive. When the chain was altered to a phenethyl moiety, the coleopteran JHE remained moderately sensitive, while the lepidopteran JHEs were much less sensitive. The lepidopteran and coleopteran JHEs did not show dramatic differences in specificity to alpha-naphthyl and rho-nitrophenyl substrates. However, as the chain length of the alpha-naphthyl substrates increased from propionate to caprylate, there was a trend towards reduced activity. The JHE of M. sexta was crystallized and the properties of the crystal suggest a high-resolution structure will follow.