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Several responses of synchronized populations of HeLa S3 cells were measured after irradiation with 220 kev x-rays at selected times during the division cycle. (1) Survival (colony-forming ability) is maximal when cells are irradiated in the early post-mitotic (G(1)) and the pre-mitotic (G(2)) phases of the cycle, and minimal in the mitotic (M) and late G(1) or early DNA synthetic (S) phases. (2) Markedly different growth patterns result from irradiation in different phases: (a) Prolongation of interphase (division delay) is minimal when cells are irradiated early in G(1) and rises progressively through the remainder of the cycle. (b) Cells irradiated while in mitosis are not delayed in that division, but the succeeding division is delayed. (c) Persistence of cells as metabolizing entities does not depend on the phase of the division cycle in which they are irradiated. (3) Characteristic perturbations of the normal DNA synthetic cycle occur: (a) Cells irradiated in M suffer a small delay in the onset of S, a slight prolongation of S, and a slight depression in the rate of DNA synthesis; the major delay occurs in G(2). (b) Cells irradiated in G(1) show no delay in the onset of S, and essentially no alteration in the duration or rate of DNA synthesis; G(2) delay is minimal. (c) Cells irradiated in S suffer an appreciable S prolongation and a decreased rate of DNA synthesis; G(2) delay is shorter than S delay.

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Inhibition with either 5-fluorodeoxyuridine or deoxyadenosine for specified periods during the division cycle of the HeLa S3 cell shows that the mid-interphase peak in sensitivity occurs just before DNA replication begins. Sensitivity subsequently decreases only after synthesis of DNA is resumed. One interpretation of the relation between fluctuations in sensitivity and in DNA synthesis is that the lethal radiation damage to these cells occurs in DNA.