The roles of microtubules and microfilaments on distribution of mitochondria were evaluated by using fluorescent staining in 2-cell embryos of golden hamsters with or without cytoskeletal assembly inhibitors. In 2-cell embryos without treatment (control), most mitochondria were accumulated at the perinuclear region, while some mitochondria were noted at the cell cortex. Microtubules were found around the nuclei, correlating with distribution of the mitochondria. In contrast, microfilaments were stained intensely beneath the cell membrane and especially at the cell-to-cell contact region. In most (82%) of embryos treated with nocodazole (an inhibitor of microtubule polymerization), mitochondria had extended into the subcortical (intermediate) region with varying degree, where they were aggregated in patches. After a treatment of cytochalasin D (an inhibitor of actin polymerization), distributional density of mitochondria decreased at the cell cortex, suggesting that mitochondria moved back around the nucleus. After a treatment of both inhibitors, the distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. Our results suggest that the translocation of mitochondria to the perinuclear region is mediated by microtubules, while the movement of mitochondria to the cell cortex is regulated by microfilaments. Microtubules and microfilaments may function as bidirectional anchors of mitochondria to the perinuclear region and to the peripheral region, respectively.