Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of approximately 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail-suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight-specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein, and cytoplasmic dynein, and 2) up-regulated expression of ubiquitin ligase genes, MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress-inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.