Translation of poliovirus RNA is initiated by entry of ribosomes into the nucleotide sequence (internal ribosomal entry site; IRES) within the 5'-untranslated region (5'-UTR). Efficiency of this translation initiation in rabbit reticulocyte lysates (RRL) was very low and was greatly enhanced by addition of the ribosomal salt-wash fraction (RSW) prepared from HeLa cells. This stimulating activity in the RSW was partially purified by gel-filtration column chromatography and its molecular weight was estimated to be higher than 240,000. Several proteins that bind specifically to the poliovirus IRES were detected in the active fraction. Among those, a 57 kDa protein, recognized by antibodies against polypyrimidide tract-binding protein (PTB), was found. In addition, La protein (52 kDa) which is a human antigen recognized by antibodies from patients with autoimmune disorders was also detected. Further purification on a hydroxylapatite column resulted in considerable loss of the stimulatory activity, accompanied by a reduction of the apparent molecular weight of active component(s). These results suggest that fully active HeLa cell stimulatory factors for the translation initiation on poliovirus RNA function in RRL as a large complex consisted of several components including PTB and La protein.