Encephalomyocarditis virus (EMCV) is a picornavirus with a worldwide distribution, capable of infecting a wide range of species. Episodes of EMCV-associated mortality have been reported in zoos and national parks around the world, including sporadic cases at Taronga Zoo, Sydney. An inactivated EMCV vaccine was evaluated by inoculating Barbary sheep (Ammotragus lervia), Indian antelope (Antilope cervicapra), Eastern wallaroos (Macropus robustus), and chimpanzees (Pan troglodytes). A proportion of the vaccinated ungulates were administered a second vaccination 4 wk after the initial dose. Neutralizing antibody titers were monitored for a period of 12 mo. One month after vaccination, all vaccinated groups had developed significant antibody titers that persisted for at least 6 mo. Animals receiving two doses of vaccine had higher titers 3, 6, and 12 mo after the initial vaccination compared with animals receiving a single vaccine dose.