Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.