Mitochondrial dysfunction is a common source of disease, affecting 1 in ~5000 individuals. The United Mitochondrial Disease Foundation states “every 30 minutes a child is born who will develop a mitochondrial disease by age 10” (www.umdf.org). The biology of mitochondria makes these problems tremendously complex. Mitochondrial function requires the coordinated expression of 37 genes encoded in mitochondrial DNA (mtDNA) inside mitochondria, and over 1000 nuclear-encoded genes whose products must be transported into mitochondria. The high mutation rate for mtDNA and the large target of nuclear genes for mutations ensures that every individual has a unique ‘mito-nuclear genotype’ that can alter fitness. Development in different environments can alter how different genotypes express adult traits. Thus, these sources of complexity are responsible for key gaps in our understanding of the genetic bases of mitochondrial disease, and more generally, the genetic variation for mitochondrial performance in natural populations. The Drosophila model we have developed provides a powerful genetic approach to dissect this complexity. We have introduced different mtDNAs into controlled nuclear genetic backgrounds and identified genetic interactions (‘mitonuclear epistases’) affecting fitness traits and gene expression. We have discovered that many of the genes with differential expression resulting from mitonuclear genetic interactions also show differential expression in response environmental perturbations. Our working hypothesis is that mitochondria integrate genetic pathways regulating changes in both the internal cellular, and external physical, environments. We will pursue three general questions. First, what signaling pathways underlie the shared gene expression responses to altered mitonuclear genotypes and altered physical environments? This will be addressed with gene expression and epigenetic experiments pairing mitonuclear genotypes and environmental stressors. Second, which nuclear genes regulate mtDNA effects on phenotypes? This will be addressed with genetic screens of the nuclear genome across a panel of variable mtDNAs. Third, do mtDNA mutations affect males more than females? The maternal inheritance of mtDNA allows direct selection in females but prevents selection in males. Male-specific deleterious mutations could accumulate in populations, a phenomenon known as Mother’s Curse. This will be addressed using sex-based phenotypic assays in a panel of mtDNA genotypes that span a range of genetic divergences. Each of these questions is relevant to current challenges in quantitative and medical genetics. The findings from this research could be informative regarding genetic questions in the identification of appropriate donors for mitochondrial replacement therapies.