We isolated two novobiocin-resistant mutants which were stable and approximately three and four times more resistant than the parent cells to novobiocin. Both mutants (Novr A2, Novr A41) were more sensitive than the wild-type cells to nalidixic acid, and cold sensitive for cell growth. When we isolated derivatives of Novr A2 and Novr A41 cells which are resistant to nalidixic acid, those are found to be phenotypically reverted to novobiocin sensitivity like wild-type cells, thereby suggesting the relationship between the targets for novobiocin and for nalidixic acid. But the cold sensitivity did not always revert to wild type, with accompanying resistance to nalidixic acid. The DNA and RNA syntheses of Novr mutants were more resistant to novobiocin but more sensitive to nalidixic acid, than those of wild-type cells. However, in vitro assays of wild-type and Novr cell extracts were unable to demonstrate any differences in the sensitivity of topoisomerase II activity to inhibition by novobiocin. While the targets of novobiocin and nalidixic acid show a mutual interaction in vivo and play a role in DNA replication and transcription, our results suggest that these targets are probably not topoisomerase II.