The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.