To elucidate the intramolecular localization of irreversible interphase phosphorylation of Physarum histone H1 [Jerzmanowski, A., & Maleszewski, M. (1985) Biochemistry 24, 2360–2367] and its effect on H1's conformational properties, the circular dichroism spectra, the pH- and salt-dependent folding, and the products of trypsin digestion for the interphase phosphorylated (with five to nine phosphates per molecule) and enzymatically dephosphorylated H1 were compared. Both phosphorylated and dephosphorylated H1 show similar amounts (6.2 and 5.5%, respectively) of helicity at high ionic strength and upon limited digestion with trypsin form identical trypsin-resistant peptides of the size slightly larger than the analogous peptide from calf thymus H1. The circular dichroism analysis of the pH-dependent folding of Physarum H1 in water shows a strong effect of phosphorylation on the folding process in both the acidic and alkaline pH region. The analysis of the products of trypsin digestion of [32P]PO4-labeled Physarum H1 before and after enzymatic dephosphorylation is consistent with the interpretation that the interphase phosphorylation occurs predominantly within the 50–70 amino acid sequence directly adjacent to the trypsin-resistant peptide on its C-terminal side and that this sequence is itself involved in some kind of loose folding at high ionic strength. The studies of the formation of the trypsin-resistant peptide (the globular domain) as a function of salt concentration show that it is induced at 300 mM lower NaCl concentration for phosphorylated than for dephosphorylated H1. These results indicate that the stable, interphase phosphorylation of Physarum HI enhances the salt-induced formation of the folded globular region in vitro. This conclusion together with our finding that only nonphosphorylated H1 occurs in the DNase I solubilized fraction of Physarum chromatin may be relevant for a mechanism of chromatin activation in Physarum.