The levels and stability of IkappaBepsilon have been examined in unstimulated and stimulated splenic B cells and compared with that of IkappaBalpha and IkappaBbeta. Primary murine splenic B cells but not T cells were found to contain high levels of IkappaBepsilon protein, equivalent to levels of the abundant IkappaBalpha. Most agents that activate IkappaBalpha and IkappaBbeta degradation do not induce rapid degradation of IkappaBepsilon. Interestingly, however, the levels of IkappaBepsilon, but not of IkappaBalpha or IkappaBbeta, are dramatically reduced upon the stimulation of B cells both in vivo and in vitro. Since IkappaBepsilon exhibits substrate specificity for NF-kappaB Rel homodimers, this suggested the possibility that changes in NF-kappaB-responsive genes might also occur during this transition. Consistent with this hypothesis, we found that a NF-kappaB reporter construct sensitive to p65/RelA homodimers is activated at the time that IkappaBepsilon levels decline following B cell stimulation. In IgG(+) B cell lines, which contain low levels of IkappaBepsilon, this same reporter construct was inactive, suggesting that the increases in Rel homodimer activity that accompany B cell stimulation are transient. However, there are differences in the level of expression of NF-kappaB-responsive genes in these IgG(+) B cell lines compared with their IgM(+) counterparts. From these data, we conclude that there are transient changes in NF-kappaB activity due to reductions in IkappaBepsilon, which might contribute to long-term, persistent changes that accompany B cell differentiation. We propose an important role for IkappaBepsilon in the differential regulation of nuclear NF-kappaB activity in stimulated B cells.