The 185 nucleotide 5' untranslated region (5'UTR) of potato virus Y ordinary strain (PVY-O) showed translation-enhancing activity on the beta-glucuronidase (GUS) gene in tobacco protoplasts. Mutational analysis of the 5'UTR was done to find sequence motifs necessary for the enhancement. Deletions within the 1-130 nucleotide region of 5'UTR stimulated the GUS expression in some cases, while the GUS activity declined with deletions in the 131-185 nucleotide region. The results indicated that the last 55 nucleotides of PVY-O 5'UTR might play the much important role in the translational enhancement in plant cells.