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% deuterium oxide (D₂O) in H₂O was administered, with the result that a low percentage of D₂O in the water increased mean life span, whereas the highest percentage used (22.5%) reduced life span. After the one-time treatment period, adult flies were maintained ad libitum with food of normal isotopic distribution. At low deuterium levels, where life span 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 high-performance liquid chromatography/mass spectrometry (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.