A suspension of freshly harvested spores of Aspergillus awamori SG1 strain was exposed to UV light. The irradiated spores with survival rate of 10%-15% were plated directly on minimal medium (MM) agar plates containing 10 mmol/L uridine and 5-fluoro-orotic acid (FOA 1 mg/mL). Five stable anti-FOA colonies with the reversed rate below 10(-9) were obtained. All anti-FOA strains, can grow on uridine or uricil-containing minimal medium agar plate but not on orotic acid-containing MM plate, indicating that the mutation happened in the URA3 gene. Northern blotting analysis showed that the mutation of URA3 gene could not be transcribed and it was confirmed by RT-PCR. With modified PEG-mediated protoplast transformation method the wild URA3 gene could complement the ura3 mutant strain SA5 to wild type. Southern blotting of the transformant indicated the wild RUA3 gene replaced the mutated ura3 gene.