The 5' untranslated region (5'UTR) of the hepatitis A virus (HAV) genomic RNA contains an internal ribosome entry site (IRES) which interacts with various cellular proteins and facilitates cap-independent translation. We report the interaction of a 25kDa protein (p25), present in certain murine tissues and most abundantly in mouse kidney, with the HAV 5'UTR. This protein was found to be a cleavage product of the polypyrimidine tract-binding protein (PTB) and competed with it for binding to the HAV 5'UTR RNA. The binding site of p25 overlapped with the reported binding site of PTB. Exogenous addition of partially purified p25 to in vitro translation reactions resulted in the inhibition of HAV IRES-mediated translation, which could be rescued by the addition of purified PTB. These results suggest that p25 is a cleavage product of PTB which binds to the HAV IRES and antagonizes the translation-stimulating activity of PTB. The presence of the 25kDa cleavage product of PTB may therefore play a role in the inhibition of HAV IRES-mediated translation in mouse tissues.