Monocytes/macrophages are key cells in the pathogenesis of human CMV (HCMV) infection, but the in vitro rate of viral production in primary human monocyte-derived macrophages (MDM) is considerably lower than in fibroblasts. Considering that the NF-kappaB signaling pathway is potentially involved in the replication strategy of HCMV through efficient transactivation of the major immediate-early promoter (MIEP), efficient viral replication, and late gene expression, we investigated the composition of the NF-kappaB complex in HCMV-infected MDMs and fibroblasts. Preliminary studies showed that HCMV could grow in primary MDM culture but that the viral titer in culture supernatants was lower than that observed in the supernatants of more permissive MRC5 fibroblasts. EMSA and microwell colorimetric NF-kappaB assay demonstrated that HCMV infection of MDMs increased p52 binding activity without activating the canonical p50/p65 complex. Moreover, Bcl-3 was up-regulated and was demonstrated to associate with p52, indicating p52/Bcl-3 complexes as the major component of the NF-kappaB complex in MDMs. Luciferase assays in promonocytic U937 cells transfected with an MIEP-luciferase reporter construct demonstrated MIEP activation in response to p52 and Bcl-3 overexpression. Chromatin immunoprecipitation assay demonstrated that p52 and Bcl-3 bind the MIEP in acutely HCMV-infected MDMs. In contrast, HCMV infection of MRC5 fibroblasts resulted in activation of p50/p65 heterodimers. Thus, activation of p52/Bcl-3 complexes in MDMs and p50/p65 heterodimers in fibroblasts in response to HCMV infection might explain the low-level growth of the virus in MDMs vs efficient growth in fibroblasts.