Purpose: Often the best method of integrating chemotherapeutic agents is unknown. Recently there has been interest in the use of combinations of the topoisomerase II inhibitors and the topoisomerase I inhibitors as these agents have shown individual activity in malignancies such as non-small-cell lung cancer. This study examined the interaction of the topoisomerase II inhibitor etoposide with the topoisomerase I inhibitor topotecan (Tpt) in V79 cells (hamster lung fibroblast cells) to determine the optimal method of delivering these agents. Methods and results: Cell survival was assessed by colony formation. Synergistic interactions were assessed by the median effect principle in which a combination index (CI) of less than one suggests a synergistic interaction. The V79 cells were exposed to sequential 24-h incubations with the two chemotherapeutic agents. Initially, equitoxic doses of the two agents were delivered (i.e. 0.0275 μg/ml of topotecan alone or 0.089 μg/ml of etoposide alone resulting in a surviving fraction of 70%; Tpt:etoposide ratio 1:3.2). It was determined that a sequence-dependent synergistic interaction(CI < 1) resulted at a lower level of cytotoxicity if the etoposide exposure followed the Tpt exposure compared to the opposite sequence. This same effect was seen after treatment of cells with various concentration (μg/ml) ratios of Tpt:etoposide (1:4.0, 1:1, 2.5:1). Conclusions: These results suggest that maximum synergy occurs for the delivery of etoposide following Tpt exposure (compared to the opposite sequence) and these findings may have important clinical implications.