The NF-kappaB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-kappaB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-kappaB is associated with the actin-binding protein alpha-actinin-4. NF-kappaB and alpha-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-alpha, alpha-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-alpha led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-kappaB co-immunoprecipitated alpha-actinin-4 from A431 cell lysates and nuclear extracts, but alpha-actinin-1 and beta-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-kappaB can bind to matrix-bound chicken gizzard alpha-actinin. We suggest that the alpha-actinin-4 is important for the NF-kappaB nuclear translocation and its functions inside the nucleus.