Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.