Initiation of the T-helper lymphocyte activation program is regulated through the T-cell receptor (TCR) and costimulatory receptors. Analysis of TCR and either anti-CD28- or interleukin 1 (IL-1)-mediated activation of the IL-2 promoter shows that costimulatory signals augment promoter activity through NF-kappaB sites. This study comparatively evaluates the mechanisms whereby signals initiated from the TCR and these two costimulatory receptors converge to synergistically increase NF-kappaB transcriptional activity. IL-1 alone stimulates an acute but transient NF-kappaB nuclear localization and a suboptimal NF-kappaB transcriptional response. In contrast, anti-CD3-anti-CD28 or anti-CD3-IL-1 synergistically stimulate prolonged NF-kappaB nuclear localization and NF-kappaB-mediated transcription. Both TCR- and costimulatory receptor-initiated synergistic NF-kappaB responses result from prolonging high rates of cytosolic IkappaB degradation during the second phase of the biphasic NF-kappaB nuclear localization. However, in contrast to previous reports, prolonged nuclear localization of NF-kappaB complexes is not necessarily associated with long-term depletion of IkappaBbeta. In response to either costimulus, c-Rel selectively translocated to the nucleus as a result of induced c-Rel expression and the continued production of c-Rel-IkappaBalpha complexes, which turn over rapidly due to the high rate of IkappaBalpha degradation in the cytosol during the second phase of the response. In contrast, IkappaBbeta is nearly completely degraded during the acute response to either IL-1 or anti-CD3-IL-1 while anti-CD3-anti-CD28 stimulates only a partial reduction (35 to 40%) in cytosolic IkappaBbeta. Cyclosporine (CsA), which inhibits stimulus-induced NF-kappaB transcriptional activity, selectively inhibits the stimulus-induced c-Rel nuclear localization and the rapid formation and degradation of c-Rel-IkappaBalpha complexes in the cytosol. CsA also inhibits both the prolonged, high rate of IkappaBalpha degradation and the lower level of IkappaBbeta turnover during the second phase of the activation response. Together, these results suggest a mechanism by which signals from the T-cell antigen receptor and either CD28 or IL-1 synergistically regulate IL-2 gene transcription by modulating NF-kappaB nuclear translocation.