Nuclear factor–kappa B (NF-κB) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-κBs, which prevent entry into the nucleus. Following cellular stimulation, the I-κBs are rapidly degraded, activating NF-κB. The active form of NF-κB rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-κB is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-κB (p65 subunit, p65-NF-κB) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-κB protein in the nucleus determined biochemically. The appearance of p65-NF-κB in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-κB can be reliably determined from IOD measurements of the immunofluorescent labeled protein.