Phosphorylated forms of Tetrahymena macronuclear histone H1 were separated from each other and from dephosphorylated H1 by cation-exchange HPLC. A homogeneous fraction of hyperphosphorylated macronuclear H1 was then used to generate novel polyclonal antibodies highly selective for phosphorylated H1 in Tetrahymena and in human cells. These antibodies fail to recognize dephosphorylated forms of H1 in both organisms and are not reactive with most other nuclear or cytoplasmic phosphoproteins including those induced during mitosis. The selectivity of these antibodies for phosphorylated forms of H1 in Tetrahymena and in HeLa argues strongly that these antibodies recognize highly conserved phosphorylated epitopes found in most H1s and from this standpoint Tetrahymena H1 is not atypical. Using these antibodies in indirect immunofluorescence analyses, we find that a significant fraction of interphase mammalian cells display a strikingly punctate pattern of nuclear fluorescence. As cells enter S-phase, nuclear staining becomes more diffuse, increases significantly, and continues to increase as cells enter mitosis. As cells exit from mitosis, staining with the anti-phosphorylated H1 antibodies is rapidly lost presumably owing to the dephosphorylation of H1. These immunofluorescent data document precisely the cell cycle changes in the level of H1 phosphorylation determined by earlier biochemical studies and suggest that these antibodies represent a powerful new tool to probe the function(s) of H1 phosphorylation in a wide variety of eukaryotic systems.