The cell cycle, oncogenic signaling, and topoisomerase (topo) IIα levels all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo IIα levels, it is difficult to assess the importance of any one of these factors independently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, therefore, developed a series of analytical procedures to individually assess the role of each of these factors during treatment with the anti-topo II drug etoposide. All studies were performed with asynchronously proliferating cultures by the use of time-lapse and quantitative fluorescence staining procedures. To our surprise, we found that neither oncogene action nor the cell cycle altered topo IIα protein levels in actively cycling cells. Only a minor population of slowly cycling cells within these cultures responded to constitutively active oncogenes by elevating topo IIα production. Thus, it was possible to study the effects of the cell cycle and oncogene action on drug-treated cells while topo IIα levels remained constant. Toxicity analyses were performed with two consecutive time-lapse observations separated by a brief drug treatment. The cell cycle phase was determined from the first observation, and cell fate was determined from the second. Cells were most sensitive to drug treatment from mid-S phase through G2 phase, with G1 phase cells nearly threefold less sensitive. In addition, the presence of an oncogenic src gene or microinjected Ras protein increased drug toxicity by approximately threefold in actively cycling cells and by at least this level in the small population of slowly cycling cells. We conclude that both cell cycle phase and oncogenic signaling influence drug toxicity independently of alterations in topo IIα levels.