Living cells are distinguished in their ability to keep the structural stability in the changing environment by means of energy supply. Understanding the mechanisms of energy conversion is impossible without knowledge of the physiological processes involved. This question is addressed to confocal laser scanning microscopy (CLSM) that has become a leader among the group of light microscopy method. The high resolution in CLSM images is reached without deconvolution procedures. Linear characteristics of its variable parameters are important for quantitative image analysis. These properties of CLSM are indispensable in the study of mitochondrial membrane potential (MMP) using fluorescent dyes. Lipophilic rhodamine dyes freely pass the plasma membrane and accumulate in the mitochondria according to electrical potential difference. CLSM images of TMRE (tetramethylrhodamine, ethyl ester)-stained U937 cells were analyzed by standard image processing procedures in ImageJ software. These procedures allow the creation of mask, applying it to original image. Average fluorescence intensity in selected regions was used as a measure of MMP changes during phytohemagglutinin treatment. This value was decreased by 34% after 2 h of lectin treatment. Some cells after mitogenic stimulation completely lost MMP. Deregulation of mitochondrial calcium handling and changes of cytoplasmic monovalent ion concentration are considered as mechanisms of MMP decrease during lymphocyte stimulation.