A restriction fragment length polymorphism (RFLP) analysis was performed on six related Acremonium strains. With respect to the restriction fragment pattern, all strains of A. chrysogenum were indistinguishable from each other but showed distinctive differences from those of A. strictum, A. flavum and Cephalosporium polyvaleurum. Using pulsed-field gel electrophoresis, we obtained different chromosome patterns from most of the Acremonium strains. Remarkably, the pattern varies in three related A. chrysogenum strains which also differ in their rate of cephalosporin C biosynthesis. The electrophoretic karyotyping was confirmed by the location of rDNA genes on separate chromosomes. Our data indicate that chromosome translocations in industrial strains may be responsible for increased beta-lactam synthesis.