During human cytomegalovirus (HCMV) infection, a series of regulated events take place following virus binding and entry into the cell, including the upregulation of cellular transcription factors, such as NF-kappa B, which play an essential role in the viral life cycle. We show here that NF-kappa B message is induced during HCMV infection and that the induction is biphasic, suggesting an initial induction at immediate-early (IE) times and a second round of induction at early times. This hypothesis is supported by experiments using cyclohexamide, which showed that the first tier of induction was drug insensitive, while the second tier was drug sensitive. We then show that virus binding alone is sufficient to stimulate NF-kappa DNA binding activity, supporting its role in the initial induction of NF-kappa B. To begin to elucidate the mechanism(s) for the second tier of NF-kappa B regulation, we examined promoter constructs from the NF-kappa B subunits (p105/p50 and p65) for responsiveness following HCMV infection. HCMV infection transactivated the p105/p50 and p65 promoters. The viral IE proteins (IE1-72, IE2-55, and IE2-86) are expressed during the time we see NF-kappa B induction, so we examined their role in NF-kappa B induction. The IE1-72, IE2-55, and IE2-86 proteins transactivated the p65 promoter, while only the IE2-55 protein transactivated the p105/p50 promoter. The p105/p50 promoter has NF-kappa B sites; therefore, upregulation could also be caused by an autoregulatory mechanism. The p65 promoter, however, has been demonstrated to contain only Sp1 sites. To investigate the potential role of SP1, we examined nuclear extracts from HCMV-infected cells. Here, we show that there is a biphasic increase in SP1 activity during viral infection and that there is apparently an absolute requirement for SP1 in the transactivation of the p65 promoter. In conclusion, we suggest a model in which the initial induction of NF-kappa B occurs through viral modulation of cellular factors and the sustained levels of NF-kappa B induction are regulated by a combination of cellular and viral factors.