Nuclear factor kappa B (NF-B)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-B remains in the cytoplasm sequestered through interaction with IB protein. Rapid translocation of NF-B from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-B in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor ␣ (TNF-␣) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-B was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-B led to a rapid increase in NF-B-associated fluorescence measured over the nucleus (F N ) concomitant with a decrease in fluorescence over the cytoplasm (F C ), which was reflected by an increase in F N /F C ratio. This rapid assay of NF-B activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-B expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules. Cytom-