Damage to DNA has been implicated in the induction of permanent cell cycle arrest or premature senescence in normal human fibroblasts. We tested the ability of a group of cancer chemotherapeutic agents or related compounds, which can cause DNA double-strand breaks (DSBs) directly or indirectly, to induce a permanent cell cycle arrest in normal proliferating fibroblasts. A brief treatment with etoposide, doxorubicin, cisplatin, or phleomycin D1 induced a block to S phase entry sustained through 15 days. Lower levels of these drugs did not induce appreciable levels of transient cell cycle arrest. Higher concentrations caused cell death that lacked the DNA degradation characteristic of apoptosis. Camptothecin, an agent that causes DNA single-strand breaks, which are converted to DSBs during S phase, was able to induce an efficient, but only transient, cell cycle arrest in these normal cells. The cells did not enter S phase until after removal of the camptothecin. These findings support the idea that permanent cell cycle arrest and cell death are typical reactions of these normal cells to drugs that can cause DSBs. In addition, we report data consistent with the concept that both actinomycin D and doxorubicin are sequestered by cells and slowly released in active form. This is consistent with the observation that both these drugs bind reversibly to intracellular components.