Abstract When the histones of metaphase and interphase Chiness hamster, HeLa S 3 and rat nephroma cells are compared by polyacrylamide gel elecrophoresis, a shift to slower mobility of metaphase F1-histone is observed. This change in mobility of the F1-histone results from enhanced phosphorylation since it is not observed after treatment of the extracted proteins with alkaline phosphatase. The phosphorylation is found in mitotic cells collected without use of a metaphase arrest agent. Further, the extent of phosphorylation of F1 is independent of the elapsed time of metaphase arrest with vinblastine. The phosphoryl-rich F1 subcomponents are dephosphorylated upon allowing the metaphase cell to enter the G 1 phase. The observed protein phosphorylation may be related to the unique physical state of metaphase chromatin. It has a pronounced influence on the chemical extraction of lysine-rich histone during isolation of metaphase chromosomes.