International concern over the potential consequences of a Bioterrorist or Biowarfare associated release of variola virus have prompted renewed interest in the vaccines for smallpox. The traditional live, replicating vaccine strains are subject to novel safety concerns associated with historical production methods in domesticated ruminants and the additional hazards that vaccinia virus poses for people with immune system abnormalities or a history of eczematous skin conditions. In this study we have examined the longevity and efficacy of immunity induced by a non-replicating smallpox vaccine candidate, modified vaccinia Ankara (MVA) in a murine model using intranasal and aerosol routes of infection. Two-step vaccinations of MVA followed by traditional Lister vaccine are compared with either Lister alone or MVA alone, and the longevity of the protection induced by MVA is assessed. MVA is found to be broadly similar to Lister. Although protection is shown to decay with time, when administered at a standard human dose the longevity of protection induced by MVA is comparable to that induced by Lister.