This study represents a detailed analysis of the structural context of the RNA polymerase I promoter of Saccharomyces cerevisiae. We determined the presence of regularly spaced nucleosomes in the non-transcribed spacer (NTS) and found that five of them have well defined positions. We show that this nucleosome positioning is restricted to the region between the 35 S and 5 S rRNA promoters, beyond which a more delocalized chromatin structure is evident. A more refined analysis detects the DNA-protein interactions on the RNA polymerase I promoter at nucleotide resolution and provides the first in vivo footprints, attributable to factors like REB1, CF, UAF and an additional protection that seems to be sensitive to the topological context. Moreover, when this analysis is extended to different growth media (YPD versus YNB), some of these protections show a growth condition dependent behaviour.