Ribosomal DNA (rDNA) genes in eukaryotes are organized into multicopy tandem arrays and transcribed by RNA polymerase I. During cell proliferation, ∼50% of these genes are active and have a relatively open chromatin structure characterized by elevated accessibility to psoralen cross-linking. In Saccharomyces cerevisiae, transcription of rDNA genes becomes repressed and chromatin structure closes when cells enter the diauxic shift and growth dramatically slows. In this study, we found that nucleosomes are massively depleted from the active rDNA genes during log phase and reassembled during the diauxic shift, largely accounting for the differences in psoralen accessibility between active and inactive genes. The Rpd3L histone deacetylase complex was required for diauxic shift-induced H4 and H2B deposition onto rDNA genes, suggesting involvement in assembly or stabilization of the entire nucleosome. The Spt16 subunit of FACT, however, was specifically required for H2B deposition, suggesting specificity for the H2A/H2B dimer. Miller chromatin spreads were used for electron microscopic visualization of rDNA genes in an spt16 mutant, which was found to be deficient in the assembly of normal nucleosomes on inactive genes and the disruption of nucleosomes on active genes, consistent with an inability to fully reactivate polymerase I (Pol I) transcription when cells exit stationary phase.