Abstract The duplication of the genes coding for 5-S RNA has been followed during the S phase of HeLa cell cultures synchronized by mitotic detechment. Replication was analyzed by exposing the cultures to bromodeoxyuridine (BrdUrd) for 3 h at successive intervals and analyzing the DNA product in CsCl density gradients. DNA containing the 5-S genes was detected in the gradients by molecular hybridization using purified 125I-labeled 5 S RNA. In addition, as an internal marker of S phase chronology, late DNA replication was followed by examining the incorporation of [ 3H]thymidine into repetitive DNA ( C 0t < 0.01 fraction ). The results indicated that >85% of the cells in the culture were synchronously dividing and that >80% of the 5-S genes replicate in the first half of S phase, whereas the rapidly reassociating fraction of the DNA replicates in the second half of S phase.