Supercoiling in eukaryotic cells can be explained by the packaging of DNA into nucleosomes in the presence of nicking-closing enzymes1. This does not, however, rule out the presence of a DNA gyrase. DNA gyrase2 is involved in the introduction and maintenance of supercoiling in prokaryotic cells3-6 and in DNA replication in Escherichia coli4,6,7. Novobiocin and coumermycin-A1 inhibit prokaryotic DNA replication4,7-11 by inhibiting the subunit of the gyrase responsible for its ATPase and energy transduction activities3,4,12-14. The inhibition of DNA synthesis in a mammalian cell15 and in rat liver mitochondria16 by novobiocin suggests that a gyrase required for initiation of replication is inhibited. Because the presence of a gyrase in eukaryotic cells would be of great interest, I have examined this question in a well characterized model system, the replication of simian virus 40 in its host (monkey) cell. The results demonstrate here that novobiocin and coumermycin A1 inhibit SV40 DNA synthesis in mammalian cells and inhibit DNA polymerase α, the replicative DNA polymerase in this system17,18. Novobiocin, but not coumermycin A1, reduces the degree of supercoiling of DNA pulse-labelled in its presence, probably through its inhibition of protein synthesis. Thus the inhibition of SV40 DNA synthesis by novobiocin does not alone prove the involvement of a DNA gyrase.