Native bulk chromatin is characterized by regular arrays of nucleosomes with defined internucleosomal distances. The nucleosome repeat length is not a constant but varies between species and cell-types, during differentiation and during gene activation. Previous studies have highlighted the importance of linker histones as a major determinant of nucleosome repeat length in vivo. We used a physiological reconstitution system derived from Drosophila embryos to study nucleosome spacing. In these extracts, histone H1 incorporation increases the apparent linker length in a gradual way. Manipulation of the chromatin assembly conditions in vitro allowed us to define additional parameters that modulate nucleosomal distances, such as protein phosphorylation events and the precise ionic conditions during the reconstitution. Interestingly, moderate changes in the concentrations of mono-, di-, and multivalent cations affect the precise distances between nucleosome cores remarkably. These changes in the ionic environment are unlikely to affect the association of linker proteins but are known to influence the folding of the nucleosomal fiber by modulation of electrostatic forces. Our results suggest electrostatic interactions in chromatin units as major determinants of nucleosome spacing. Nucleosome spacing and the folding of the nucleosomal fiber can therefore be explained by common principles, most notably the neutralization of charges in linker DNA.