A small inhibitor RNA (IRNA) isolated from yeast has previously been shown to efficiently block poliovirus and hepatitis C virus IRES-mediated translation by sequestering mammalian RNA-binding (transacting) factors that play important roles in cap-independent translation. Here we have investigated the IRNA-binding proteins that might be involved in cap-independent translation in the yeast Saccharomyces cerevisiae. We have identified Zuotin, a DnaJ chaperone protein similar to mammalian HSP-40 chaperone, which interacts strongly with IRNA. Using ZUO1-deleted S. cerevisiae, we demonstrate a preferential requirement of Zuo1p for cap-independent translation mediated by the 5' untranslated region of the yeast TFIID mRNA. Further studies using zuo1delta S. cerevisiae complemented with various Zuo1p mutants indicate that the DnaJ domain of Zuo1p, known to influence its interaction with HSP-70, significantly affects cap-independent translation. These results demonstrate for the first time a role for an established chaperone protein in cap-independent translation of a cellular mRNA.