We have examined critically whether or not new and old histones mix in the octameric units of nucleosomes during chromatin replication. MH-134SC cells were density-labeled by culturing with amino acid mixtures enriched with dense isotopes 2H, 13C, and 15N. Mononucleosomes obtained from labeled cells were fractionated by rate zonal sedimentation through a sucrose gradient in heavy water (Senshu et al. (1985) Eur J. Biochem. 150, 575-580). The fractionation can be performed under conditions that do not destabilize nucleosomes. Density-labeling yielded heterogeneous mononucleosome species which showed higher sedimentation rates than normal mononucleosomes. However, they were indistinguishable with respect to their protein compositions, electrophoretic mobilities, electrophoretic patterns of single-stranded DNA fragments liberated by DNase I digestion, electrophoretic mobilities and sedimentation velocities of the DNA moieties, and metabolic stabilities of the histone moieties. These data suggest that the heterogeneity of density-labeled mononucleosomes resulted from the formation of histone octamers density-substituted to different degrees. This would be an inevitable consequence of mixing of new and old histones in the octameric unit of nucleosomes.