Abstract—–Because of conflicting reports on the phototoxicity of rhodamine 123 (Rh 123), we have undertaken a study of Rh 123 photosensitization in severalin vitrosystems. First, Rh 123 is not a photodynamic agent and does not react with singlet oxygen. Second, when bound to cytochrome c (Cyt c), Rh 123 photosensitizes ferro Cyt c but not ferri Cytcdegradation by an oxygen‐independent process. When delivered to skin fibroblasts where it specifically stains mitochondria, Rh 123 photosensitizes membrane damage. These results are consistent with the hypothesis that Rh 123 is a phototoxic stain. The lack of photosensitivity of Rh 123‐stained mitochondria in some cell lines might therefore be due to specific structural features.