The photochemotherapeutic effect of the mitochondria-specific dye rhodamine-123 (Rh-123) on human glioma cells in culture was studied. Cultured U-251MG glioma cells were incubated for 30 minutes in 10 micrograms/ml of Rh-123 and then exposed to blue-green light between 488 and 514.5 nm using a continuous-wave argon laser. Cells that were treated with Rh-123 and the argon laser at power densities less than 200 mW/sq cm demonstrated increasing tumor-cell killing with increasing time of exposure to laser light. Tumor-cell killing achieved with power densities of light less than 200 mW/sq cm was shown to be due solely to a photochemical effect and not to a direct (thermal) effect of the laser. The photochemical effect was dependent upon the intracellular concentration of Rh-123 and the length of light exposure, and not the intensity of light. The selective retention of Rh-123 by glioma cells and its exclusion from normal cells in conjunction with its photoactivated cytotoxicity suggest that Rh-123 may be a useful photosensitizing drug for the treatment of malignant gliomas in situ.