Upon lytic infection of permissive cells, the herpes simplex virus (HSV) transactivator protein VP16 associates with an accessory protein termed host cell factor (HCF). Binding to HCF activates VP16 for association with the octamer motif-binding protein Oct-1, to form a multiprotein-DNA complex responsible for activating transcription of the HSV immediate early genes. We show that HCF comprises a series of related polypeptides that range from 110 to 300 kd, all of which are encoded by a single gene. Although there is no obvious sequence similarity between HCF and other known proteins, HCF contains eight repeats of a new 26 amino acid motif. cDNAs encoding HCF predict a large open reading frame of 2035 codons. When expressed in human cells, this large open reading frame encodes both the 300 kd and smaller HCF polypeptides, indicating that the smaller polypeptides arise by processing of the 300 kd protein.