DNA analysis of Cephalosporium acremonium revealed that this important fungus for biotechnology contains, apart from chromosomal DNA, two types of extrachromosomal DNA which may be useful for the development of a homologous vector system: 1. Plasmid-like (pl) DNA consisting of circular molecules, but of rather heterogeneous buoyant density and restriction pattern. In contrast to Podospora pl DNA it is not associated with mitochondrial DNA. 2. Mitochondrial (mt) DNA consisting of the smallest circular molecules (26.7 kb) known so far in filamentous fungi. It contains unique cleavage sites for at least 3 restriction endonucleases. A detailed physical map of mtDNA was constructed. A genetics map was also established by hybridization with rho(-) DNA of Saccharomyces cerevisiae. Thus sufficient information for the construction of a mitochondrial vector is now available.