Reactivation of the human polyomavirus BK (BKV) has emerged as an important cause of allograft rejection in renal transplant recipients. Expression of the viral early promoter that leads to production of T-antigen is the first event in viral lytic infection. In an effort to understand the mechanism involved in the activation of BKV early gene (BKV(E)) expression, we analyzed the promoter/enhancer region of the virus and identified binding motifs for the inducible transcription factors NF-kappaB and C/EBPbeta, which are in juxtaposition to each other downstream from the early gene transcription initiation site. Results from transfection studies demonstrate that overexpression of the p65 subunit of NF-kappaB, but not C/EBPbeta stimulates transcription of the BKV(E) promoter in CV-1 cells. Interestingly, low level expression of C/EBPbeta showed a synergistic effect on p65 activation of the BKV(E) promoter, suggesting a functional cooperativity between these two regulators upon viral gene transcription. Results from DNA-binding studies showed the ability of p65 and C/EBPbeta to bind independently with BKV DNA as removal of the binding site for p65 or C/EBPbeta had no significant effect on the interaction of p65 and C/EBPbeta with their motifs, respectively. Functional evaluation of the mutant promoter with no binding sites for either NF-kappaB or C/EBPbeta showed that the observed synergism requires the p65 but not the C/EBPbeta binding site, suggesting cross-talk between C/EBPbeta and p65 in this event. Results from the co-expression of p65 and C/EBPbeta showed no evidence for the formation of a DNA-protein complex containing both p65 and C/EBPbeta, although results from protein-protein interaction studies verified the ability of C/EBPbeta to interact with p65. A dominant-negative isoform of C/EBPbeta which contains the DNA binding but not activation domain of full-length C/EBPbeta cooperated with p65 in activating the BKV(E) promoter, suggesting a functional interaction between the b-ZIP domain of C/EBPbeta and NF-kappaB.