Point mutations were introduced at random into cDNA corresponding to nucleotides 260-833 of the encephalomyocarditis virus (EMCV) 5' noncoding region. This region contains the internal ribosome entry site (IRES). The mutations were identified by sequence analysis and the effect on the activity of the IRES was determined using in vitro translation reactions in rabbit reticulocyte lysate. Significantly defective mutants each contained multiple point mutations. These mutants were constructed into a dicistronic mRNA expression plasmid and the activities of the mutant IRES elements were determined using the vaccinia virus/T7 RNA polymerase transient expression system in vivo. The most severely defective of these mutants displayed about 5% of wild-type activity. The activities, relative to wild type, of these mutant IRES elements determined using in vitro and in vivo assays were similar. Two deletion mutants, lacking sequences from the 5' terminus to nt 411 and 484, were also constructed. Each of these deletions inactivated the IRES in vivo (to less than 1% of wild-type activity). Coexpression within cells of the wild-type EMCV IRES, either alone or linked to another coding sequence, enhanced the activity of each of the defective IRES elements except that deleted to nt 484. The results are consistent with a model in which different regions of the IRES participate in a discontinuous transfer of an initiation complex to the 3' end of the IRES element for initiation of protein synthesis to occur.