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Yeasts with different types of energy metabolism were examined with fluorescent microscopy in the presence of tetramethylrhodamine methyl ester in order to investigate the role of mitochondria in the development of a defense response to heat treatment. Heat shock was found to increase the electrochemical polarization of the inner mitochondrial membrane in an obligate aerobe Debaryomyces vanrijiae. In D. vanrijiae yeasts grown on a glucose medium, the addition of azide, 2,4-dinitrophenol, and antimycin A during “mild” heat shock treatment suppressed the development of induced thermotolerance and inhibited synthesis of a heat shock protein Hsp60. In a facultative anaerobe Saccharomyces cerevisiae grown on a glucose medium (fermentative energy metabolism), antimycin A had no effect on the development of induced thermotolerance and accumulation of a heat shock protein Hsp104. However, after growing S. cerevisiae on ethanol-containing medium under conditions of effective oxidative metabolism, antimycin A blocked the fulfillment of the defense program and prevented the enhanced synthesis of Hsp104. The results provide evidence that mitochondria play a principal role in the development of stress response and support the notion that activated transcription of stress genes depends on changes in electrochemical polarization of the inner mitochondrial membrane.