NF-κB proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-κB activation and input into the regulation of gene activity have been comprehensively investigated, its cytoplasmic functions are poorly understood. In this study we addressed effects of the compartmentalisation of NF-κB proteins RelA/p65 and p50 in relation to the inhibitor IκB-α, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-κB family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NF-κB was active or not. Sub-cellular fractionation demonstrated that the proportion of RelA/p65 differed in diverse sub-cellular fractions, and that the cytoskeleton harboured about 7% thereof. Neither the nuclear nor the cytoskeleton fraction did contain IκB-α. The cytoskeleton binding of RelA/p65 and p50 was further confirmed by co-localisation and electron microscopy data. During 30-min EGF stimulation similar dynamics were found for RelA/p65 and IκB-α in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IκB-α in the membrane. Furthermore, EGF stimulation for 30 min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-κB are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IκB-α. Moreover, we discovered the existence of a dynamic, IκB-α-free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-κB stimulation.