HCF-1 is a mammalian protein required for cell proliferation. It is also involved in transcriptional activation of herpes-simplex-virus immediate-early gene transcription in association with the viral transactivator VP16. HCF-1 and a related protein called HCF-2 possess a homologue in Caenorhabditis elegans that can associate with and activate VP16. Here, we demonstrate developmental regulation of C. elegans HCF (CeHCF) phosphorylation: a hyperphosphorylated form of CeHCF is present in embryos, whereas a hypophosphorylated form is present in L1 larvae. The phosphorylation patterns of endogenous CeHCF in worms and ectopically synthesized CeHCF in mammalian cells are remarkably similar, suggesting that the way CeHCF can be recognized by kinases is conserved in animals. Phosphorylation-site mapping of endogenous CeHCF, however, revealed that phosphorylation occurs at four clustered sites in the region of the protein that is not highly conserved among HCF proteins and is not required for VP16-induced complex formation. Indeed, phosphorylation of either CeHCF or human HCF-1 appears to be dispensable for association with VP16. All four CeHCF phosphorylation sites match the consensus recognition site for the cell-cycle kinases CDC2 and CDK2. Consistent with this similarity and with the developmental phosphorylation of CeHCF in C. elegans embryos, CeHCF phosphorylation is cell-cycle-regulated in mammalian cells.