The sensitivity of HeLa S3 cells to 220 kv X-rays was measured in terms of cell survival (colony development) during the G2 phase of the cell generation cycle, employing two procedures designed to free G2 cultures from contaminating cells from other phases of the cycle. Treatment of synchronous cultures (obtained initially by mitotic selection) with high specific activity tritiated thymidine (HSA-(3)HTdR) selectively eliminated S phase cells, while addition of vinblastine permitted removal of cells as they entered mitosis. It was found that HeLa S3 cells become increasingly sensitive as they progress through G2. The pattern of sensitivity fluctuations observed in synchronous HeLa S3 populations selected by the foregoing method was compared with that found in synchronous cultures prepared by the HSA-(3)HTdR method of Whitmore. The latter method had been used previously with mouse L cells, which were found to undergo a different pattern of sensitivity fluctuations. The two methods yield similar results for HeLa cells in the S and G2 phases of the cycle. It may be concluded, therefore, that the discrepancies between HeLa and mouse L cells do not arise from methodological factors, but represent fundamental differences between the cell types.