Low concentrations of novobiocin are toxic to permeable yeast cells, but do not inhibit type II topoisomerase activity. Furthermore, the enzyme does not bind specifically to novobiocin-Sepharose. These observations are in agreement with genetical analyses. Mutations at a single locus that confer novobiocin resistance and temperature sensitivity exhibit a similar phenotype to cells treated with novobiocin, but are not topoisomerase II mutants.