DNA supercoiling is a major regulator of transcription in all organisms. This process is regulated by type I and type II DNA topoisomerases that are targets for microbial antibiotics and/or anti-tumour drugs. Despite extensive studies in this field, no information is available on the response of Bacillus (B) species to eukaryotic DNA topoisomerase inhibitors. Here we found that B. thuringiensis BMG1.7 and HD9 strains are sensitive to DNA gyrase inhibitors (e.g. ciprofloxacin, novobiocin) and to etoposide VP16, a specific inhibitor of eukaryotic type II DNA topoisomerase. Inhibitory or sub-inhibitory concentrations of VP16 induced a drug-tolerant response: an immediate inhibition of growth, followed by a prolonged (10-12 h) lag in growth, and then resumption of normal growth subsequent to overnight culture. Inhibition of the DNA gyrase ATPase activity in B. subtillis 168 by novobiocin activated 80 genes and repressed 89 genes at 20 min after drug addition (P<0.05). The altered genes belonged to various functional categories dominated by those whose products maintained DNA integrity, mediated transport of low molecular-weight compounds, and SOS response genes. Quantitative RT-PCR revealed comparable effects of novobiocin in B. subtillis and thuringiensis on gene expression. In contrast to novobiocin, VP16 treatment resulted in a moderate effect on gyrA, gyrB, and topA gene expression. However, dinB and lexA genes, involved in SOS response, displayed relatively high transcriptional levels compared to those seen in untreated cells. Furthermore, a small DNA plasmid isolated from novobicin-treated B. thuringiensis BMG 1.7 contained a ladder of partially relaxed topoisomers, while the electrophoretic mobility of its counterpart isolated from VP16-treated cells was unchanged. Collectively, the present study would assist in defining common and/or different pathways affected by eubacterial and eukaryotic type DNA topoisomerase inhibitors in the same living organism.