In this study, the potential of ozone in inactivating enterovirus 71 (EV71) free particles was investigated using either various ozone flow rates of 100, 80 or 60 mg/h or a constant flow rate of 80 mg/h, given to culture medium or various pH culture media containing EV71, respectively. Results demonstrated that EV71 inactivation by ozone was related to the kinetics of ozone solubility, approximately 99% inactivation being obtained in the exponential phase of ozone solubility. However, the inactivation rate was dependent on the ozone input flow rate and positively enhanced at acidic pH. Inactivation of intracellular EV71 was also studied. At a constant ozone supply of 60 mg/h, a significant reduction of intracellular virus titer (> or =99%, p < 0.01) was obtained after 45 or 60 min exposure but with low cell viability. Upon 30 min exposure, however, 45% cell viability was retained. The results indicate that the inactivating effect of ozone on intracellular EV71 virus is dependent on exposure duration.