Oxidative stress is one of the major factors underlying mitochondrial dysfunctions. One of the most promising approaches for alleviating or preventing oxidative stress is the use of cationic uncouplers that accumulate in mitochondria in accordance to the level of the membrane potential, producing "mild" uncoupling. Based on this theoretical background, cationic rhodamine 19 butyl ester (C4R1) was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these tests were presented (Khailova et al. (2014) Biochim. Biophys. Acta, 1837, 1739-1747), but one publication could not accommodate all data on interactions of C4R1 with isolated mitochondria. In addition to previously presented data, we found that the effect of C4R1 on the rate of oxygen uptake is subject to temporal variations, which probably reflects variable rates of C4R1 entry into the mitochondria. Consequently, transient stimulation of respiration can be followed by inhibition. C4R1 was found not to shunt electron flow from complex I of the respiratory chain; it largely acted as an inhibitor of complex I in the respiratory chain and showed antioxidant activity. C4R1 taken at low, non-uncoupling concentrations enhanced the uncoupling activity of fatty acids (e.g. palmitate). Relatively low C4R1 concentrations stimulated opening of a nonspecific Ca2+/Pi-dependent pore. ATP synthesis and hydrolysis were substantially inhibited by C4R1 at low concentrations that had no appreciable effects on respiration in states 4 and 3 and only slightly decreased the membrane potential. Besides, conditions were revealed allowing correct evaluation of the membrane potential generated at the inner mitochondrial membrane with safranin O upon oxidation of both succinate and NAD-dependent substrates in the presence of C4R1.