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We previously reported that DNA topoisomerase II (topo II) is required for the G0-to-S phase transition in mammalian cells [Hossain et al. (2002) ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells. Genes Cells 7, 285–294]. In this study, we examined whether the requirement for topo II is evolutionarily conserved in Drosophila and yeast. ICRF-193, a catalytic inhibitor of topo II, inhibited DNA synthesis in Drosophila Schneider cells released from the G0 (stationary) phase, whereas the drug did not inhibit DNA synthesis in Schneider cells released from the M phase. Depletion of topo II mRNA by RNA-interference (RNAi) in G0-phase Schneider cells resulted in significant inhibition of DNA synthesis after release from G0-arrest. In the yeast topo II temperature-sensitive (ts) mutant, the initial cycle of DNA synthesis occurred at a restrictive temperature after release from starvation-induced G0 phase and doubling of the DNA content in the cells was confirmed by both flow cytometry and fluorescence spectrophotometry. DNA synthesis in yeast cells after release from the G0 phase was also observed in the presence of ICRF-193. Doubling of the DNA content was observed during spore germination of topo II ts mutant yeast at a restrictive temperature as determined by fluorescence spectrophotometry. These results indicate that topo II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast.