Rhodamine 123 is a specific fluorescent probe for mitochondrial localization in living cells. It improves mitochondrial detection compared to conventional light microscopy. This dye allows the visualization of mitochondrial distribution changes following Rous sarcoma virus transformation and mitochondrial shape changes after colchicine treatment. Mitochondria are dynamic organelles that show plasticity, mobility, and morphological diversity. Their morphology is influenced by the cell's metabolic state, cell cycle, development, differentiation, and pathological conditions. Mitochondrial morphological and functional changes are associated with neoplastic transformation. Although mitochondria in fixed cells have been studied extensively, less attention has been given to those in living cells. Previous studies were limited by the lack of high-resolution visualization techniques. Janus Green B, a relatively specific mitochondrial dye, aids in recognition but causes mitochondrial distortion and cytotoxic effects. Rhodamine compounds have been used as histological stains, and various fluorescent probes have been used to study mitochondrial energy state. Rhodamine 6G has been shown to inhibit oxidative phosphorylation and block adenine nucleotide translocase in isolated rat liver mitochondria. It has also been proposed that the phenotypic expression of rhodamine 6G resistance in yeast mutants may be controlled by nuclear and mitochondrial or cytoplasmic genes. Rhodamine 123 stains mitochondria directly without passage through endocytic vesicles and lysosomes, providing low-background, high-resolution fluorescent images without apparent cytotoxic effects. The dye was tested with various concentrations, times, and temperatures to determine optimal conditions for staining. The results show that rhodamine 123 is a specific fluorescent probe for mitochondria in living cells. It was used to study mitochondrial organization in Rat 1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus and in IMR-33 gerbil fibroma cells treated with colchicine. The results indicate that rhodamine 123-stained structures are mitochondria. The antibiotic valinomycin, which induces mitochondrial swelling, also caused swelling of rhodamine 123-stained structures and release of rhodamine 123 fluorescence into the cytoplasm. Rhodamine 123 is an unusual rhodamine derivative that produces green fluorescence when excited at 485 nm and red fluorescence when excited at 546 nm. The chemical structure of rhodamine 123 is shown in Figure 3. Mitochondria stained with rhodamine 123 at concentrations of 0.1, 1.0, 10, 100, and 1000 µg/ml showed no significant difference in staining intensity at 100 and 1000 µg/ml compared to 10 µg/ml. Although some toxic effects were observed at higher concentrations, there was no apparent cytotoxicity at 10 µgRhodamine 123 is a specific fluorescent probe for mitochondrial localization in living cells. It improves mitochondrial detection compared to conventional light microscopy. This dye allows the visualization of mitochondrial distribution changes following Rous sarcoma virus transformation and mitochondrial shape changes after colchicine treatment. Mitochondria are dynamic organelles that show plasticity, mobility, and morphological diversity. Their morphology is influenced by the cell's metabolic state, cell cycle, development, differentiation, and pathological conditions. Mitochondrial morphological and functional changes are associated with neoplastic transformation. Although mitochondria in fixed cells have been studied extensively, less attention has been given to those in living cells. Previous studies were limited by the lack of high-resolution visualization techniques. Janus Green B, a relatively specific mitochondrial dye, aids in recognition but causes mitochondrial distortion and cytotoxic effects. Rhodamine compounds have been used as histological stains, and various fluorescent probes have been used to study mitochondrial energy state. Rhodamine 6G has been shown to inhibit oxidative phosphorylation and block adenine nucleotide translocase in isolated rat liver mitochondria. It has also been proposed that the phenotypic expression of rhodamine 6G resistance in yeast mutants may be controlled by nuclear and mitochondrial or cytoplasmic genes. Rhodamine 123 stains mitochondria directly without passage through endocytic vesicles and lysosomes, providing low-background, high-resolution fluorescent images without apparent cytotoxic effects. The dye was tested with various concentrations, times, and temperatures to determine optimal conditions for staining. The results show that rhodamine 123 is a specific fluorescent probe for mitochondria in living cells. It was used to study mitochondrial organization in Rat 1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus and in IMR-33 gerbil fibroma cells treated with colchicine. The results indicate that rhodamine 123-stained structures are mitochondria. The antibiotic valinomycin, which induces mitochondrial swelling, also caused swelling of rhodamine 123-stained structures and release of rhodamine 123 fluorescence into the cytoplasm. Rhodamine 123 is an unusual rhodamine derivative that produces green fluorescence when excited at 485 nm and red fluorescence when excited at 546 nm. The chemical structure of rhodamine 123 is shown in Figure 3. Mitochondria stained with rhodamine 123 at concentrations of 0.1, 1.0, 10, 100, and 1000 µg/ml showed no significant difference in staining intensity at 100 and 1000 µg/ml compared to 10 µg/ml. Although some toxic effects were observed at higher concentrations, there was no apparent cytotoxicity at 10 µg