Potassium leakage is one of the first events that appear after photosensitization of red blood cells. This event may subsequently lead to colloid osmotic hemolysis. The aim of our study was to determine which photodynamically induced damage is responsible for increased membrane cation permeability. This was done by studying the effect of dimethylmethylene blue (DMMB)-mediated photodynamic treatment (PDT) on different membrane transport systems. Inhibition of band 3 activity (anion transport) showed a comparable light dose dependency as PDT-induced potassium leakage, whereas glycerol transport activity was inhibited only at higher light doses. Dipyridamole (DIP), an inhibitor of anion transport, protects band 3 against DMMB-induced damage, and prevents the increase in cation permeability of the membrane. Damage to glycerol transport was partially reduced when PDT was performed in the presence of DIP. Because DIP has no affinity for the glycerol transporter, this protection might result from the reduced photodamage to band 3. These results support the hypothesis that band 3 might be involved in glycerol transport. Glucose transport was not affected by DMMB-mediated PDT. The present results are the first to show a causal relationship between DMMB-mediated photodamage to band 3 and increased cation permeability of red blood cells.