Mitochondrial genetic variation can have profound effects on fitness, and the mitotype must interact with both the nuclear genes and the environment. We used Drosophila to investigate the extent to which mitotype effects on lifespan and activity are modulated by nucleotype and environmental variation. When nucleotype is varied, mitochondrial effects on lifespan persisted but were relatively small, and still male biased. Varying food as well, mitotype had substantial effects on male climbing speed, modifiable by nucleotype but less so by diet. Finally, mitotype affected fly lifespan much more in a cage environment compared with a vial, also modifiable by nucleotype and diet. The cage may represent a stressful environment. Mitochondrial genotype may affect fitness much more in conditions of stress, which may have implications for human health.