In this study, a sensitive high performance thin-layer chromatography method (HPTLC) was developed to detect lipids in microgram (ug) quantities of hemolymph. The effect of age, diurnal rhythms, and flight activity on hemolymph 1,2-diacylglyceride concentrations in male and female Heliothis zea (Boddie) (Lepidoptera: Noctuidae) were quantitated. The HPTLC method involved a two-stage, one dimensional solvent system, 1 ). benzene:ether(diethyl):ethanol:acetic acid, 50:40:2.0:0.2; 2). hexane:heptane: ether (diethyl): acetic acid, 63:18:18:1. The solvent system separated alkanes, steryl ester, mono-, di- and triacylglycerides, free fatty acids and cholesterol. Lipids were detected with a fluorescent reagent, 1 mM 6-p-toludino-2-napthalenesulfonic acid (TNS) in 95% methanol. Detection sensitivities were 50 ng qualitatively and 500 ng quantitatively. Most male and female Heliothis zea flight activity occurred between 17⁰⁰ and 3⁰⁰ EST each day. Male flight activity after the day of eclosion through 6 days was 0.8, 1.0, 7.0, 3.3, 3.8, 2.8 and 4.0 times the flight activity of the females. Females had a unimodal pattern of flight activity between 17⁰⁰ and 19⁰⁰ Males had a bimodal pattern between 17⁰⁰ and 19⁰⁰, but another higher peak of flight activity existed between 20⁰⁰ and 24⁰⁰. The hemolymph of Heliothis zea males and females prior to flight (17⁰⁰) was composed of mainly diacylglycerides (50% w/w), and triacylglycerides (35% w/w). In addition, cholesterol esters (2%) and less than 1% monoacylglycerides and cholesterol were present, but free fatty acids were not detectable ( <0.5 ug/ul). The lipid class, 1,2-diacylglycerides, apparently are used for flight energy. They occured in the highest concentrations in both males and females prior to flight (17⁰⁰) (approximately 32 ug/ul), then decreased steadily throughout flight to approximately 16 ug/ul at 2⁰⁰ when flight activity was reduced or ceased. Males and females appear to have similar 1,2-diacylglyceride concentrations prior to, during and after flight activity. / M.S.