The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains of Drosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 184.108.40.206) activity in all of the strains. Dietary ethanol also increased the incorporation of 14C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of 14C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids.