Cowpox is an orthopoxvirus infection endemic in European wild rodents, but with a wide host range including human beings. In this longitudinal study we examined cowpox in two wild rodent species, bank voles Clethrionomys glareolus and wood mice Apodemus sylvaticus, to investigate the dynamics of a virus in its wild reservoir host. Trapping was carried out at 4-weekly intervals over 3 years and each animal caught was uniquely identified, blood sampled and tested for antibodies to cowpox. Antibody prevalence was higher in bank voles than in wood mice and seroconversion varied seasonally, with peaks in autumn. Infection was most common in males of both species but no clear association with age was demonstrated. This study provides a model for studying other zoonotic infections that derive from wild mammals since other approaches, such as one-off samples, will fail to detect the variation in infection and thus, risk to human health, demonstrated here.