HCF-1 is a highly conserved and abundant chromatin-associated host cell factor required for transcriptional activation of herpes simplex virus immediate-early genes by the virion protein VP16. HCF-1 exists as a heterodimeric complex of associated N- (HCF-1(N)) and C- (HCF-1(C)) terminal subunits that result from proteolytic processing of a precursor protein. We have used small-interfering RNA (siRNA) to inactivate HCF-1 in an array of normal and transformed mammalian cells to identify its cellular functions. Our results show that HCF-1 is a broadly acting regulator of two stages of the cell cycle: exit from mitosis, where it ensures proper cytokinesis, and passage through the G(1) phase, where it promotes cell cycle progression. Proteolytic processing is necessary to separate and ensure these two HCF-1 activities, which are performed by separate HCF-1 subunits: the HCF-1(N) subunit promotes passage through the G(1) phase whereas the HCF-1(C) subunit is involved in proper exit from mitosis. These results suggest that HCF-1 links the regulation of exit from mitosis and the G(1) phase of cell growth, possibly to coordinate the reactivation of gene expression after mitosis.