Abstract An electrophoretic molecular karyotype has been established for an industrial strain of Cephalosporium acremonium using transverse alternating field electrophoresis. Eight chromosome bands were detected in gently prepared DNA samples. The size of the chromosomes ranged from approx. 1700 kb up to greater than 4000 kb. The total genomic content for this strain of C. acremonium is at least 22 500 kb. Hybridization analyses revealed that two key genes involved in cephalosporin C biosynthesis are not physically linked to one another. The isopenicillin N synthetase gene ( pcbC) resides on chromosome (chr.) VI while the deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase gene ( cefEF) resides on chr. II. The ribosomal RNA genes were located on chr. VII, while the β-isopropylmalate dehydrogenase gene ( LEU2) was found to be linked to the pcbC gene on chr. VI.