Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria‐localized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F‐actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3‐butanedione 2‐monoxime and the sulphydryl‐modifying agent N‐ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F‐actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.