The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector when it is administered 30 min prior to exposure of Chinese hamster ovary K1 cells to radiation (i.e., a dose modification factor of 1.4) at a concentration of 4 mM. Under these exposure conditions, topoisomerase (Topo) I and II alpha activities and associated protein contents were measured in cells of the K1 cell line using the DNA relaxation assay, the P4 unknotting assay and immunoblotting, respectively. WR-1065 was ineffective in modifying Topo I activity, but it did reduce Topo II alpha activity by an average of 50%. The magnitude of Topo II alpha protein content, however, was not affected by these exposure conditions. The effects on the cell cycle were monitored by the method of flow cytometry. Exposure of cells to 4 mM WR-1065 for up to 6 h resulted in a build-up of cells in the G2/M-phase compartment. However, under these conditions and in contrast to Topo II inhibitors used in chemotherapy, WR-1065 is an effective radioprotective agent capable of protecting against both radiation-induced cell lethality and mutagenesis. One of several mechanisms of action attributed to aminothiol compounds such as WR-1065 has been their ability to affect endogenous enzymatic reactions involved in DNA synthesis and repair and progression of cells through the phases of the cell cycle. These results are consistent with such a proposed mechanism and demonstrate in particular a modifying effect by WR-1065 on Topo II, which is involved in DNA synthesis.