We aimed to investigate the dynamics of the NF-kappaB signaling pathway in living cells using GFP variants of p65-NF-kappaB, IkappaBalpha, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-kappaB inducing kinase (NIK) and IkappaB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IkappaBalpha enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-kappaB and IkappaBalpha revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795-805, which counteracts the inherent NLS at amino acids 143-149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-kappaB.IkappaB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-kappaB.