In anticipation of the development of a vaccine against cytomegalovirus (CMV), we conducted a large, nationally representative serosurvey to examine the seroprevalence of CMV in Australia. Sera were collected opportunistically from laboratories around Australia. Age- and gender-representative samples were tested for CMV antibody. The population-weighted rate of CMV seropositivity in subjects between 1 and 59 years of age was 57% (95% confidence interval, 55.2 to 58.6%). An association between CMV seroprevalence and increasing age was recognized; however, little overall difference in seroprevalence between the sexes was found. The finding that high levels of CMV exposure occur in the first few years of life suggests that for a universal vaccination program to have maximal impact, the vaccine would need to be delivered to infants and have a long duration of protective efficacy. This is the first national serosurvey looking at cytomegalovirus in the Australian community. This study provides valuable information that can be used to examine the incidence of infection in the community and help focus the administration of a future CMV vaccine to appropriate target populations.