The cytotoxic action of 5-aza-2'-deoxycytidine (5-AZA-CdR) in synchronized cells and logarithmic- and plateau-phase cultures of EMT6 murine tumor cells was investigated. 5-AZA-CdR produced a greater cell kill of S phase cells than of cells in G1 phase. Cells in the logarithmic phase of growth were more sensitive to the cytotoxic effects of 5-AZA-CdR than cells in the plateau phase of growth. These results indicate that 5-AZA-CdR produces a preferential kill of cells in the S phase of the cell cycle. 5-AZA-CdR did not block the cell cycle progression of cells into S phase. The survival curve of EMT6 cells exposed to 5-AZA-CdR suggests that the cytotoxic action of this analogue is not self-limiting. The mutagenic activity of 5-AZA-CdR was investigated using induction of 6-thioguanine resistance in Chinese hamster ovary cells. 5-AZA-CdR was not a detectable mutagen in this assay system.