In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels.