Mechanism for the photoinduced increase in the lysosomal K permeability is still unknown. In this study, we investigated the effect of photodamage-induced membrane rigidification on the lysosomal K permeability by measuring the membrane potential with bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol and by monitoring proton leakage with p-nitrophenol. Membrane fluidity was measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Methylene blue–mediated photodamage to lysosomes decreased their membrane fluidity and increased their K permeability. The photoinduced increase in the K permeability can be reversed by fluidizing the rigidified lysosomal membranes with benzyl alcohol. The results suggest that the membrane rigidification induced by photodamage may increase lysosomal K permeability. This conclusion is supported by the observation that rigidifying lysosomal membranes by the treatment with membrane rigidifier cholesteryl hemisuccinate also enhanced the lysosomal K permeability.