4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and other DNA intercalating agents produce protein-associated DNA strand breaks, the formation of which are mediated by topoisomerase-like chromosomal proteins. As topoisomerases would be expected to be most active during DNA replication, DNA synthesis inhibitors may alter the sensitivity of cellular DNA to intercalator-induced scission. We report that treatment of L1210 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) (0.1 microM) or hydroxyurea (HU) (0.1 mM) for 18 hr resulted in a 2- to 2.4-fold enhancement of m-AMSA-induced protein-associated DNA single-strand breaks and DNA-protein cross-links as measured by alkaline elution. This enhancement was dependent on the duration of ara-C or HU treatment as well as on the concentration of ara-C or HU. Enhancement did not correlate with any alteration in cellular uptake of intercalator or with ara-C- or HU-induced alterations in the DNA synthetic rate. The DNA within nuclei isolated from ara-C- or HU-treated cells also displayed an enhanced susceptibility to m-AMSA-induced scission. There was a correlation between enhanced single-strand break formation and recruitment of cells into S-phase as well as between single-strand break formation and the production of a hypomethylated state of cellular DNA. Concurrent with the enhancement of m-AMSA-induced cellular DNA effects was a synergistic effect on m-AMSA cytotoxicity by ara-C or HU. This enhancement of intercalator effects was also found for the intercalator Adriamycin. We propose that these sublethal concentrations of ara-C and HU alter chromatin structure possibly via DNA hypomethylation and/or altered DNA-histone interactions so that intercalator-induced DNA effects are enhanced. Alternatively, the topoisomerase-like activity involved in intercalator-induced, protein-associated DNA break production may be increased in the nuclei of ara-C- or HU-treated cells.