DNA amplification as a mechanism to increase gene expression has been established as a cause of cytotoxic drug resistance and appears to play a role in tumor cell progression. In order to investigate factors which control the process of DNA amplification we have been using a simian virus 40 (SV40)-transformed Chinese hamster cell line (CO60) as a model system. This cell line can be induced to amplify integrated viral DNA with a variety of agents. In this report the following is shown. (a) Addition of ethacridine, an intercalative compound, or ethanol to the culture media inhibits amplification induced by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine or by gamma-irradiation in a dose-dependent fashion. In the case of N-methyl-N'-nitro-N-nitrosoguanidine induction (50 microM), the highest concentrations of ethacridine (40 microM) or ethanol (2% v/v) tested reduced SV40 amplification from about 20-fold to less than 2-fold. (b) Neither substance induces significant amplification when applied alone over a wide range of concentrations (0.01-20 microM ethacridine; 0.001-2% ethanol). (c) Significant inhibition of amplification is achieved with nearly nontoxic concentrations of both substances (10 microM; 1%), (d) Without direct interference with the inducer. It is concluded that ethacridine or ethanol treatment uncouples the toxic effects of an alkylating agent or ionizing radiation from their ability to induce amplification in CO60 cells.