We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.