We have investigated the effects of brief, non-specific deuteration of Drosophila melanogaster by including varying percentages of 2H (D) in the H2O used in the food mix consumed during initial development. Up to 22.5% D2O in H2O was administered, with the result that a low percentage of D2O in the water increased mean lifespan, while the highest percentage used (22.5%) reduced lifespan. After the one-time treatment period, adult flies were maintained ad libitum with food of normal isotopic distribution. At low deuterium levels, where lifespan extension was observed, there was no observed change in fecundity. Dead flies were assayed for deuterium incorporation by complete hydrolysis in hot 12 N HCl solution followed by subsequent HPLC/MS. Isoleucine and leucine residues showed a small, linear dose-dependent incorporation of deuterium at non-exchangeable sites. Although high levels of D2O itself are toxic for other reasons, higher levels of deuterium incorporation, which can be achieved without toxicity by strategies that avoid direct use of D2O, are clearly worth exploring.