High resolution gel electrophoresis was used to demonstrate an inverse relationship between catenation linking number and superhelicity in highly catenated simian virus 40 chromosomes caused by exposure to the topoisomerase II inhibitor ICRF-193. Since ICRF-193 does not unwind DNA, we conclude that the decreased superhelicity in catenated SV40 daughter chromosomes is a direct result of increased catenation. It is likely that catenation decreases superhelicity by interfering with the formation of nucleosomes. The absence of normal chromatin structure in regions of catenation may facilitate access to topoisomerase II under normal conditions. ICRF-193 does not prevent initiation of SV40 DNA replication.