Abstract: Encephalomyocarditis virus (EMCV) can infect a wide range of vertebrate species including swine and non-human primates, but few data are available for humans. We therefore wanted to gain further insight into the mechanisms involved in EMCV infection of human cells. For this purpose, we analyzed the permissiveness of primary human cardiomyocytes towards two strains of EMCV; a pig myocardial strain (B279/95) and a rat strain (1086C). In this study, we show that both strains productively infect primary human cardiomyocytes and induce complete cytolysis. Binding and infection inhibition experiments indicated that attachment and infection are independent of sialic acid and heparan sulfate for B279/95 and dependent for 1086C. Sequence comparison between the two strains and three-dimensional analysis of the capsid revealed that six of the seven variable residues are surface-exposed, suggesting a role for these amino acids in binding. Moreover, analysis of variants isolated from the 1086C strain revealed the importance of lysine 231 of VP1 in the attachment of EMCV to cell-surface sialic acid residues. Together, these results show a potential for EMCV strains to use at least two different binding possibilities to initiate infection and provide new insights into the mechanisms involved in primary human cell recognition by EMCV.