Internal ribosome entry sites (IRES) were first identified as sequences in viral genomes that allow cap-independent translation initiation. The IRES sequence from the encephalomyocarditis virus (EMCV) is able to direct internal translation initiation that allows co-expression of multiple genes from one mRNA. Although EMCV-IRES has also been demonstrated to be functional in transgenic plants, the characteristics of the EMCV-IRES in plant cells have not been fully defined. Using a transient expression system by microprojectile bombardment and quantitative reporter gene assay, we have analyzed EMCV-IRES activity in plant cells. We show that the spacing between the IRES sequence and the second cistron is crucial for IRES-dependent translation efficiency. However, the order of cistrons is not an important factor for the EMCV-IRES-dependent translation in plant cells. Studies in different plant species reveal species specificity for EMCV-IRES activity. Analysis of transgenic tobacco plants suggested that the EMCV-IRES-dependent translation is suppressed in roots.