Histone H1(0), a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1(0) content. These observations suggest that H1(0) may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1(0) decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1(0) in the nucleus. We defined new protocols to analyze H1(0) content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1(0) and the position of cells in the cell cycle showed that no significant difference in H1(0) content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1(0) and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1(0) and that the decrease of H1(0) is not a prerequisite of cell division. We propose that the accumulation of H1(0) is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.