Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [3H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak approximately 12 hr following medium change; beta interferon inhibits the enhanced uptake of [3H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and approximately 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [3H]-thymidine, measured either at 22 degrees C or 37 degrees C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22 degrees C, the initial rate of thymidine transport at a high (500 microM) thymidine concentration, determined within the first 30 sec of [3H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37 degrees C, labeled precursors accumulate in acid-soluble material for approximately 8 min after the addition of [3H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [3H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.