The rates of DNA synthesis were determined for each of two consecutive journeys through S-phase by highly synchronized HeLa cells. Cells at various times after release from the metabolic block were pulsed with [3H]thymidine. The amount of radioactivity in whole cells, purified DNA, and Okazaki fragments provided indexes of the rates of DNA synthesis. Measurements of the average DNA content per cell by the diphenylamine method and the individual DNA content per cell by DNA:propidium fluorescence provided better estimations of the actual rates of DNA synthesis, independent of thymidine metabolism. Unsynchronized cells that had been pulsed with [3H]thymidine were sorted into early, middle, and late S-phase preparations for estimations of the amount of radioactivity per cell. There were differences in the rates predicted by each of the various methods. Rates estimated by fluorescence measurements of DNA content per cell, or by diphenylamine measurements of average DNA content per cell exhibited a pattern of an initial burst, followed by a decreased rate then a final burst. Similar patterns were obtained for the amount of radioactivity in Okazaki fragments, and in early, middle, and late S-phase cells separated from a log-phase culture by electronic cell sorting. Rates estimated by measurements of the amount of radioactivity in whole cells, and the specific activity of purified DNA exhibited a different pattern of an initial slow rate, followed by a maximal rate then a slow rate.