The role of NF-kappaB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-kappaB response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEP-enhancer that, although this region is essential for HCMV growth, none of the four NF-kappaB response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.