Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.