Rat erythrocytes incubated in photoactivated riboflavin system in the presence of serum caused a rapid hemolysis. During the course of illumination, a marked efflux of intracellular K⁺, increase in osmotic fragility and promotion of lipid peroxidation of the cell occurred prior to hemolysis indicating that riboflavin-induced photohemolysis is colloidosmotic process. In the absence of serum, however, no hemolysis was observed at any time of incubation, suggesting that membrane damages are enhanced by the illumination in the presence of serum. Furthermore, the illumination of cells in the photoactivated riboflavin system in the presence of serum under continued anaerobic conitions did not exhibit any significant hemolysis. These results indicate that the photohemolysis of cells is due to oxidative damages of cell membrane that initiated te hemolysis. Singlet oxygen (¹O₂) scavengers employed in this study strongly inhibited the photohemolysis, suggesting that cells are damaged oxidatively by ¹O₂ generated in photoactivated riboflavin system in the presence of serum. Furthermore, triplet quenchers and α-tocopherol suppressed this photohemolysis. A reference was made concerning a possible adverse effect of riboflavin on infant erythrocytes in phototherapy.