Translational inhibition of most mRNAs and preferential translation of mRNAs coding heat shock proteins (Hsps) occur in most cells under heat shock stress. For most Hsp mRNAs, preferential translation in heat-shocked cells is conferred by their 5'-untranslated regions (5'-UTRs). However, the preferential translation directed by 5'-UTRs during heat shock remains mostly unknown in plants. Here, we found that the mRNA of Hsp81-3, which is an Arabidopsis Hsp90 family gene, continued to be associated with polysomes in heat-shocked Arabidopsis suspension-cultured cells. The Hsp81-3 5'-UTR was found to contribute to the efficient translation of capped reporter mRNAs in heat-shocked Arabidopsis protoplasts using a transient expression assay. Further characterization of the Hsp81-3 5'-UTR revealed that the anterior half of the 5'-UTR is important for the efficient translation in heat-shocked protoplasts. Moreover, the Hsp81-3 5'-UTR was highly capable of enhancing translation from uncapped reporter mRNAs relative to the 5'-UTR of a housekeeping gene in both normal and heat-shocked protoplasts. These Hsp81-3 5'-UTR-directed translations both in capped and uncapped reporter mRNAs were substantially reduced by the insertion of an upstream AUG at the 5'-end of the 5'-UTR, indicating that ribosomes are recruited to the 5'-end of the Hsp81-3 5'-UTR regardless of temperature and the presence or absence of the cap structure. These results suggest that the preferential translation of Hsp81-3 mRNA in heat-shocked Arabidopsis cells involves a ribosome scanning from the 5'-end of the 5'-UTR rather than ribosome entry to the internal site.