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Lipophorin is the major hemolymph protein responsible for lipid transport between tissues of insects. Lipophorins from several insect species in order Diptera (the fruit fly Drosophila melanogaster from the suborder Brachycera, the mosquito Aedes aegypti; the phantom midges Chaoborus maximus and minimus; the black fly Simulium vittatum; the crane fly Nephrotoma abbreviata, all from the suborder Nematocera) were isolated and characterized. All lipophorins consisted of two protein subunits of approximately 240 and 75 kDa each. The density of each lipophorin was in the high-density lipoprotein range (1.112 to 1.128 g/ml). The predominant neutral lipid carried by lipophorin from insects belonging to the infraorder Culicomorpha was triacylglycerol. Lipophorin from the crane fly Nephrotoma abbreviata, which belongs to the infraorder Tipulomorpha, carried approximately equivalent amounts of diacylglycerol and triacylglycerol. Lipophorin from D. melanogaster was found to carry diacylglycerol as the predominant neutral lipid.