We demonstrate that the 5' untranslated region ( 5' UTR) plays an important role in determining translation efficiency in Aspergillus oryzae, using a model beta-glucuronidase (GUS) expression system. Alterations in the 5' UTR resulted in an increase in GUS activity of up to eightfold, without affecting mRNA levels. Moreover, using the most effective 5' UTR construct, we could achieve remarkable intracellular overproduction of GUS protein; and the GUS level reached more than 50% of the total soluble protein. This is the first experimental evidence indicating the feasibility of improving recombinant protein yield by promoting translation initiation in filamentous fungi.