Sera from 12 different species of Hawaiian mammals and birds as well as from humans were examined by plaque reduction neutralization test for antibodies against encephalomyocarditis virus (EMC). The highest prevalence of antibodies was found among rats (Rattus sp.), mongooses, pigs, and cows. Lower rates were observed among humans, cats, and wild birds. The prevalence of EMC antibodies among R. rattus on four Hawaiian islands ranged from 8–86%, indicating considerable geographic variation in virus activity. Sera from selected rodent populations in Colorado, the Caroline and Philippine islands, Panama, and Iran were also tested and the results confirmed the association of EMC with members of the genus Rattus. Laboratory-reared R. rattus, R. norvegicus, and Mus musculus were fed three low-passage EMC virus strains. The frequency of infection among the exposed animals varied with rodent species and with each virus strain, suggesting differences in species susceptibility as well as in strain infectivity. Blood, urine, and stool specimens were collected from the animals for virus isolation at regular intervals after virus administration. With the exception of single blood and fecal samples collected 24 h post-feeding, no virus was recovered from any of the specimens from experimentally infected rodents. Attempts to demonstrate contact transmission among EMC-infected and control mice caged together were also negative. Infection of birds (pigeons and chickens) with EMC following parenteral inoculation was demonstrated. EMC virus did not survive in mosquitoes after intrathoracic injection.