Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 degrees C followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 degrees C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 degrees C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho-) and in cells with disrupted SCO1 gene (Deltasco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O2-) into the cells. Accumulation of O2- and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho-, Deltasco1), or in cells pretreated with O2- scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.