Previously we showed that infection of human type II airway epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in the cytoplasm by association with the IkappaB inhibitor and can be released only following IkappaB degradation. In this study, we examined the spectrum of IkappaB isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In contrast to the rapid and robust activation of RelA DNA binding that peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-alpha), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak until 24 h after infection. A549 cells expressed the IkappaB inhibitory subunits IkappaBalpha, IkappaBbeta, and p105; however, following either stimulus, only the IkappaBalpha and IkappaBbeta steady-state levels declined in parallel with the increase in RelA DNA-binding activity. The >120-min half-life of IkappaBalpha in control cells was shortened to 5 min in TNF-alpha-stimulated cells and to 90 min in pRSV-infected cells. Although IkappaBalpha was resynthesized within 30 min following recombinant human TNFalpha treatment due to a robust 25-fold increase of IkappaBalpha mRNA expression (the RelA:IkappaBalpha positive feedback loop), following pRSV infection, there was no reaccumulation of IkappaBalpha protein, as infected cells produced only a 3-fold increase in IkappaBalpha mRNA at 24 h, indicating the RelA:IkappaBalpha positive feedback loop was insufficient to restore control IkappaBalpha levels. IkappaBalpha proteolysis induced by TNF-alpha occurred through the 26S proteasome, as both 26S proteasome activity and IkappaBalpha proteolysis were blocked by specific inhibitors lactacystin, MG-132, and ZLLF-CHO. Although total proteasome activity in 24-h pRSV-infected lysates increased twofold, its activity was >90% inhibited by the proteasome inhibitors; surprisingly, however, IkappaBalpha proteolysis was not. We conclude that RSV infection produces IkappaBalpha proteolysis through a mechanism primarily independent of the proteasome pathway.