Pretreatment of the human lymphoblastoid cell line CCRF-CEM with 0.02 microM arabinosyl cytosine (ara C) enhances both the cytotoxic and the DNA-damaging effects of etoposide. This concentration of ara C is itself non-cytotoxic and results in no detectable DNA damage as measured by alkaline elution. Ara C pretreatment results in the synchronisation of cells, a 24-h pretreatment resulting in the accumulation of cells in the early S phase. The sensitivity of cells to etoposide-induced cytotoxicity was increased 2.5 times and DNA damage was enhanced 1.66 times by this pretreatment. Maximal potentiation of etoposide-induced DNA damage (2.06-fold increase) was observed after 48 h continuous treatment with ara C, but no further enhancement of cytotoxicity occurred. Cell-cycle analysis demonstrated that 48 h ara C treatment resulted in the accumulation of cells in the late S/G2M phase. Cells returned to a normal cell-cycle distribution within 24 h of the removal of ara C, and the potentiation of etoposide activity was then reduced to a 1.3- to 1.4-fold level. DNA damage induced by etoposide following ara C pretreatment was qualitatively identical to that produced by etoposide alone, suggesting a mechanism involving topoisomerase II. To investigate this possibility, we measured topoisomerase II protein levels by immunoblotting. Measurement of topoisomerase II levels in whole-cell lysates of ara C-pretreated cells showed a 3- to 5-fold increase in topoisomerase levels relative to total protein content. This suggests that elevated enzyme levels may be responsible for the increased sensitivity of ara C-pretreated cells to etoposide.