Despite the importance of human noroviruses (NoVs) in public health, little information concerning the effectiveness of ozone against NoVs is available. We determined the efficacy of ozone disinfection using murine norovirus (MNV) as a surrogate of human NoV. MNV in ozone demand-free buffer was exposed to a predetermined dose of ozone at two different pHs and temperatures. The virus remaining in the solution was analyzed by plaque assay, real-time TaqMan reverse transcriptase PCR (RT-PCR) (short template), and long-template conventional RT-PCR. Under all conditions, more than 99% of the MNV was inactivated by ozone at 1 mg/liter within 2 min. Both RT-PCR assays significantly underestimated the inactivation of MNV, compared with that measured by plaque assay. Our results indicate that NoV may be more resistant to ozone than has been previously reported. Nevertheless, proper ozone disinfection practices can be used to easily control its transmission in water.Noroviruses (NoVs) are the major etiological agent of gastroenteritis in all age groups worldwide and are transmitted via a fecal-oral route (3,18,22,27,35). As important waterborne and food-borne pathogens, they are included on the U.S. EPA Contaminant Candidate List 3 (CCL 3) (7, 11). Water can be contaminated by NoV-containing feces released from either symptomatic or asymptomatic patients (26). High concentrations of the virus in fecal matter of infected people (ϳ10 9 viral particles/g of feces) (25), low infectious doses (Ͻ10 viral particles) (32), and high resistance to various environmental stresses (9, 21) contribute to the high prevalence of outbreaks caused by NoV. Proper disinfection of drinking and recreational water is necessary to prevent waterborne outbreaks (33).Among currently available analytical methods of virus cultivation, the plaque assay is the most convenient and commonly applied analytical method for determining virus inactivation by a disinfectant (6, 12). However, no conventional cultivation assays or convenient laboratory animal models have been available for human NoV. Thus, despite the importance of NoV in public health, limited information is available regarding the effectiveness of various disinfectants against NoV.Ozone (O 3 ), a strong oxidizing agent, has been widely used as a disinfectant in drinking water treatment plants in many European countries, receiving more attention after the discovery of potentially harmful chlorine by-products (37, 38). Owing to its high effectiveness and lack of residue after disinfection, ozone can be used for both surface and groundwater disinfection. Previously, a number of ozone disinfection studies of enteric viruses such as adenoviruses and enteroviruses have been reported (29,33). In all of these studies, ozone disinfection was determined by either plaque assay of feline calicivirus (FCV) or long-template (LT) reverse transcriptase PCR (RT-PCR) assay of human NoV. FCV, a member of the Caliciviridae family, is easily cultivated in vitro and has been commonly used as a surrogate of NoV (10, ...