The poor bioenergetic state in mitochondria containing mtDNA with the 4977-bp deletion has been well documented. However, information on mitochondrial reactive oxygen species (ROS) generation at rest or under intense oxidative stress in mitochondria lacking the 4977-bp mtDNA fragment inside intact living cells was insufficient. We used cybrids containing truncated mtDNA lacking the 4977-bp fragment and measured ROS levels inside cybrids by fluorescence probe, 2',7'-dichlorodihydrofluorescein (DCF), and confocal microscopy. Mitochondrial ROS at resting state was slightly higher in cybrids containing 4977-bp deletion mtDNA as compared to cybrids without mtDNA defects. For intense oxidative stress treatment, cybrids were treated with 5 mM H2O2 for 10 min. Consecutive DCF images were acquired after H2O2 had been washed away. Progressive increase of DCF signals, especially in the mitochondrial area, was observed in cybrids containing 4977-bp deletion mtDNA, even long after the brief, intense H2O2 treatment. This result suggests that a feed-forward, self-accelerating vicious cycle of mitochondrial ROS production could be initiated in cybrids containing 4977-bp deletion fragment mitochondria after brief, intense H2O2 treatment. This mechanism may play an important role in the pathophysiology of the disease process caused by mitochondria containing mtDNA with the 4977-bp deletion.