The cycle-dependent cytotoxicity of seven chemotherapy agents was compared with the use of subpopulations of Chinese hamster ovary cells separated into the various phases of the cell cycle by centrifugal elutriation. The proportion of cells killed by either beta-cytosine arabinoside or hydroxyurea agreed well with the proportion of S-phase cells in the treated subpopulations. Cell-cycle survival patterns were also determined for bleomycin (BLM), adriamycin (ADR), cis-diamminedichloroplatinum (II) (cis-DDP), Asaley, and Yoshi 864. Three of the agents (cis-DDP, Asaley, and Yoshi 864) had similar cell-cycle survival patterns in that they all preferentially killed cells in the G1 phase. In contrast, BLM preferentially killed cells in G2+M phase, whereas ADR was most cytotoxic to cells in middle to late S-phase. This investigation demonstrated that separation of cultured mammalian cells into the various cell-cycle phases by centrifugal elutriation allows the rapid determination of the phase-dependent cytotoxic effects exerted by chemotherapy agents.