Phosphorylation of histone H3 (H3) on Ser‐10 correlates with chromatin condensation at mitosis. A new monoclonal antibody (anti‐H3‐P) was developed that recognizes phosphorylated H3 (H3‐P). This antibody was used in multiparameter flow cytometric analysis to relate H3 phosphorylation in individual human leukemic cells to the cells' position in the cycle as well as their expression of cyclins A and B1. Mitotic cells, from prophase to telophase, reacted with anti‐H3‐P; the binding of the antibody to chromatin of interphase cells was several times weaker. Cell growth in the presence of staurosporine, an inhibitor of the kinase(s) that phosphorylate H3, abolished the cells' reactivity with the antibody. The reactivity also was abolished by incubation of permeabilized mitotic cells with alkaline phosphatase. These data indicate that, within permeabilized cells, the antibody is indeed specific for H3‐P and does not detect the unphosphorylated epitope. All cells reacting with anti‐H3‐P, with the exception of prophase and early prometaphase, were cyclin A negative; the expression of cyclin B1 in these cells was threefold higher than in G₂ cells. The analysis of phosphorylation of H3 in individual cells when combined with multiparameter analysis of their cycle position and expression of other proteins offers new possibilities to study molecular mechanisms associated with the G₂ to M transition and chromatin condensation. It also offers an assay to screen in vivo inhibitors of kinase(s) or phosphatase(s) involved in H3 phosphorylation or dephosphorylation, and it provides a valuable marker to identify mitotic cells by cytometry. Cytometry 32:71–77, 1998. © 1998 Wiley‐Liss, Inc.