The purpose of this study was to investigate the mechanism of direct current (DC)-induced cytotoxicity. To test the working hypothesis that electrolysis products are responsible for the DC-induced cytotoxicity, the cytotoxic effects between the direct and indirect DC treatment against human polymorphonuclear cells (PMNs) was compared. The indirect DC treatment (treatment with the culture medium exposed to DC) was comparable in cytotoxicity to the direct DC treatment, suggesting that electrolysis products have an important role in DC-induced cytotoxicity. Metal ions released from different electrodes into the culture medium were quantified by the inductively coupled plasma-mass spectroscopy. Higher concentrations of Ag, Zn, and Ni and chromium were released from Ag, Zn, and stainless steel (St) electrodes, respectively, whereas much lower concentrations of Ni and Ti were released from Ni-Ti electrode. Further, electron spin resonance spectroscopy with spin-trapping agent showed that the direct current with the following metal electrodes generated alkoxyl radical (St and Ni-Ti electrodes), hydrogen radical (Ag and Au electrodes), and both carbon and alkoxyl radicals (Zn electrode), respectively. These results suggest that free radicals and metal ions released from electrodes contribute to the cytotoxicity of DC treatment used for iontophoresis.