Nuclear factor-kappa B (NF-kappaB) plays an important role in UV-induced skin tumorigenesis. Activation of NF-kappaB by UV-irradiation is composed of two phases. The early phase culminates with maximal levels of DNA binding ability at 4 h postirradiation and is dependent on translational inhibition. The late-phase activation of NF-kappaB occurs between 16 and 48 h post-irradiation and the mechanism is not clear due to the fact that NF-kappaB was activated in the presence of high level of IkappaBalpha. In this report, we provide evidence that without translational inhibition, the transcription of IkappaBalpha was induced by UV-irradiation. In the late-phase of UV-induced NF-kappaB activation, the IkappaBalpha depletion is the combined result of regulation at both transcriptional and translational levels. Neither ubiquitination nor proteasomal degradation have detectable attributions to IkappaBalpha breakdown. We also demonstrate that UV only induced phosphorylation of p65(S276), while tumor necrosis factor-alpha induced phosphorylation at both Ser276 and 536 sites of p65. Based upon our results, we propose a novel mechanism for translation-regulated IkappaBalpha depletion and MSK-mediated NF-kappaB activation at 24 h post-UV-irradiation.