The possibility of selective killing of transformed cells in a mixed population of untransformed and transformed cells was examined using a cell culture system of rat 3Y1 fibroblasts (parental 3Y1 cells, 3Y1 cells transformed with either SV40, polyoma virus, Rous avian sarcoma virus, E1A gene of adenovirus type 12, or H‐v‐ ras oncogene). The principle of the selective killing is as follows. Under suboptimal culture conditions, untransformed cells are inhibited from progressing through G1 phase and retain viability, while transformed cells are not arrested. When DNA synthesis is inhibited for a long period, both types of cells in S phase die. Therefore, if we administer inhibitors of G1 progression and of DNA synthesis simultaneously to a cell population consisting of untransformed and transformed cells, most untransformed cells are arrested in G1 phase, retaining viability, while transformed cells leak from the G1 phase, cease DNA synthesis, and gradually die. The present study shows that all types of transformants in stationary‐phase cultures (consisting of cells mainly with a G1 DNA content) were killed to higher extents compared with untransformed cells, during incubation at lower cell densities with a combination of cycloheximide (G1 inhibitor) and aphidicolin (DNA‐synthesis inhibitor). However, cycloheximide reduced the killing effect of aphidicolin by changing the irreversible DNA‐synthesis inhibition to a reversible inhibition. The availability of G1 inhibitors that do not interfere with the irreversibility of inhibition of DNA synthesis is required for the treatment of cancer based on this idea.