The transcriptional control region of the human neurotropic polyomavirus JC virus contains a consensus NF-kappa B site which has been shown to enhance both basal and extracellular stimulus-induced levels of transcription of JC promoters. Here, we show that the expression of JC late promoter constructs containing the NF-kappa B site is decreased by cotransfection with the NF-kappa B/rel subunits, p50 and p52, but enhanced by the p65 subunit. However, JC promoter constructs lacking the NF-kappa B site were activated by p52 and p50 and repressed by p65. This antithetical response of the JC promoter mapped specifically to the D domain, which is a target site for the cellular transcription factor, YB-1. Band shift studies indicated that YB-1 and p65 modulate each other's binding to DNA: YB-1 augments the affinity of p65 for the NF-kappa B site, while p65 reduces the binding of YB-1 to the D domain. Results from coimmunoprecipitation followed by Western blot (immunoblot) analysis suggest an in vivo interaction between p65 and YB-1 in glial cells. Functionally, YB-1 appears to act synergistically with p65 to control transcription from the NF-kappa B site. A converse pattern is seen with the D domain, in which YB-1 acts synergistically with p50 and p52 to regulate transcription. p50 and p52 may function as transcriptional activators on the D domain by removing the repressive effect of p65 on YB-1 binding to the D domain. On the basis of these data, we propose a model in which NF-kappa B/rel subunits functionally interact with consensus NF-kappa B sites or YB-1-binding sites, with disparate effects on eukaryotic gene expression.