Acremonium chrysogenum, a producer of cephalosporin C, was subjected to DNA-mediated transformations using a Vector without bacterial DNA sequences. Recombinant fungal strains were generated with a gel-purified DNA fragment, carrying only the mutated P-tubulin gene from A. chrysogenum. The lack of any bacterial DNA was verified by Southern hybridization analysis and polymerase chain reaction amplifications to detect even residual DNA sequences. This procedure can be referred to as a self-cloning experiment for which less restricted working regulations are needed. Finally, the transfer of a synthetic hirudin gene by cotransformation demonstrated that any DNA molecule can be introduced into the A. chrysogenum genome without bacterial marker genes. This seems to be highly relevant for biotechnical processes in which safe recombinant producer strains are required to satisfy governmental restrictions.