A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrdUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, Lobs, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of X-rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions Lobs continues to increase only until operating replicons have completed their replication. This value for Lobs then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before X-irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 micron, whereas for Chinese hamster ovary cells, the average replicon size is about 42 micron.