Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1, NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1. The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.