The distribution of unphosphorylated and phosphorylated isoforms of linker histone H1 protein was examined during the cell cycle of HeLa cells by quantitative light and electron microscopic immunocytochemistry. Immunolabeling with a monoclonal antibody directed against the globular domain of H1 (anti-H1), which recognized predominantly unphosphorylated H1, and a polyclonal antibody directed against hyperphosphorylated H1 (anti-H1P) revealed that: (1) H1 immunolabeling was lowest at the start of S phase (S(S)), and then increased progressively during the middle (S(i)) to end of S phase (S(e)), mitosis (M) and telophase (T) to reach the highest level in G1 phase, at which time there was a sudden reduction in H1 immunolabeling before the start of S phase; (2) H1P immunolabeling paralleled this progressive increase, but only until M phase, after which it abruptly disappeared and was virtually absent in G1; (3) H1P immunolabeling in S and M phase was found on both nuclear chromatin or chromosomes and in the cytoplasm, while H1 immunolabeling was found only on nuclear chromatin or chromosomes where it was predominantly localized on condensed chromatin. Our study indicates that H1 dissociates from the DNA to a large extent during replication and chromosome condensation, but not in interphase when cells are transcriptionally active.