Drosophila simulans is known to harbor three distinct mitochondrial DNA (mtDNA) haplotype groups (siI, -II, and -III) with nearly 3.0% interhaplotypic divergence but <0.06% intrahaplotypic diversity. With the large amount of genetic variation in this system, the potential power to detect intraspecific fitness differences in fly lines that carry distinct haplotypes is great. We test three life-history traits on fly lines with known sequence differences in the mtDNA genome after controlling the nuclear genome by backcrossing. We find that flies with the siI haplotype are fastest developing and have the lowest probability of surviving to three experimental periods (2-6, 12-17, and 34-39 days of age). Wild-type males with siIII mtDNA were more active while disruption of specific coadapted nucleo-mitochondrial complexes caused a significant decrease in activity. These results are discussed in the context of the geographic distribution of each haplotype.