We have examined the relationship of phosphate content in histone H1 of Physarum polycephalum to mitotic chromosome condensation and affinity for deoxyribonucleic acid (DNA). H1 undergoes a series of posttranslational phosphorylations which increase its apparent molecular weight on NaDodSO₄-polyacrylamide gels. Our studies confirm the observation by Bradbury and co-workers [Bradbury, E. M., Inglis, R. J., Matthews, H. R., & Sarner, N (1973) Eur. J. Biochem. 33, 131-139; Bradbury, E. M., Inglis, R. J., & Matthews, H. R. (1974) Nature (London) 247, 257-261] that the accumulation of phosphate in H1 increases markedly shortly before the onset of mitosis. However, we show in pulse-chase experiments with both ³²PO₄H1 and [¹⁴C]lysine H1 that there is no significant dephosphorylation of the histone either during or shortly after mitosis, suggesting that nonspecific postmitotic dephosphorylation of H1 is not a prerequisite for chromosome decondensation. We also show that both phosphorylated and unphosphorylated forms of H1 bind with somewhat greater affinity to single-stranded DNA-cellulose than to native DNA-cellulose and that phosphorylation weakens the affinity of H1 to both forms of DNA-cellulose.