In eukaryotic cells, DNA is organized into arrays of repeated nucleosomes where the shorter nucleosome repeat length (NRL) types are associated with transcriptionally active chromatin. Here, we tested a hypothesis that systematic variations in the NRL influence nucleosome array folding into higher-order structures. For NRLs with fixed rotational settings, we observed a negative correlation between NRL and chromatin folding. Rotational variations within a range of longer NRLs (188 bp and above) typical of repressed chromatin in differentiated cells did not reveal any changes in chromatin folding. In sharp contrast, for the shorter NRL range of 165-177 bp, we observed a strong periodic dependence of chromatin folding upon the changes in linker DNA lengths, with the 172 bp repeat found in highly transcribed yeast chromatin imposing an unfolded state of the chromatin fibre that could be reversed by linker histone. Our results suggest that the NRL may direct chromatin higher-order structure into either a nucleosome position-dependent folding for short NRLs typical of transcribed genes or an architectural factor-dependent folding typical of longer NRLs prevailing in eukaryotic heterochromatin.