DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.