Transcription factor NF-kappa B is a central regulator of inflammatory, immune and acute phase reactions. It rapidly initiates the transcription of a wide variety of target genes in response to various pathogenic stimuli. Because NF-kappa B is predominantly controlled at a posttranscriptional level through association with the inhibitory I kappa B subunits, its activation cannot be monitored directly at a cellular level by means of detecting new mRNA or protein synthesis. In this study, we describe a monoclonal antibody, designated alpha-p65MAb, that recognizes an epitope which includes the nuclear location signal (NLS) of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-kappa B. alpha-p65MAb recognized human and rodent p65 only when I kappa B alpha was not bound to p65. Thus, the IgG3 selectively stained the activated, nuclear form of NF-kappa B in cultured cells. Unlike I kappa B, the MAb and its Fab fragments did not inhibit the DNA binding activity of NF-kappa B in mobility shift assays. We show that alpha-p65MAb is suitable to study the activation state of NF-kappa B in cryosections of tissues.