Resistance to topoisomerase I (TOP1)-targeting drugs such as topotecan often involves upregulation of topoisomerase II (TOP2), with accompanying increased sensitivity to TOP2-targeting drugs such as etoposide. This trial was designed to investigate sequential topoisomerase targeting in the treatment of patients with high-risk acute myelogenous leukemia. An initial cohort of patients received topotecan and cytosine arabinoside daily for 5 days. Serial samples of circulating mononuclear cells were examined to evaluate peak elevations of TOP2-alpha protein expression. In subsequent cohorts, etoposide was administered daily for 3 days, beginning 6 h after initiation of the topotecan infusion. The etoposide dose was escalated to determine a maximum-tolerated dose. Circulating mononuclear cells were analyzed for TOP1 mutations and ABCG2 protein expression. In addition, systemic and intracellular topotecan concentrations were measured. Thirty-one patients were enrolled. On the basis of TOP1-alpha protein levels in three patients with peripheral blast counts greater than 50%, etoposide administration began 6 h after initiation of the topotecan/cytosine arabinoside infusion. Using this schedule of administration, the maximum-tolerated dose of etoposide was 90 mg/m. No TOP1 mutations were identified, but increases in ABCG2 expression during the infusion were observed in mononuclear cells from two of four evaluable patients. Administration of etoposide 6 h after initiation of a topotecan/cytosine arabinoside infusion is feasible and is associated with clinical activity. Analysis of TOP2-alpha protein levels in this small number of patients indicated that peak increases occurred earlier than expected based on earlier publications. Upregulation of ABCG2 was detected in circulating cells and may represent an inducible form of drug resistance that should be investigated further.