The immunosuppressant FK506 activates NF-kappaB through IkappaBalpha degradation in nonlymphoid cells. In the present study, we analyzed mechanisms by which FK506 induces IkappaBalpha degradation. We found that FK506 induces the degradation of both IkappaBalpha and IkappaBbeta and that the time courses of the FK506-induced degradation are quite different from degradation induced by interleukin 1 (IL-1). Despite this difference, FK506-induced IkappaBalpha degradation was dependent on the N-terminal Ser-32 and Ser-36 phosphorylation sites and was mediated by proteasomes, as is the case for IL-1-induced IkappaBalpha degradation. We further showed that FK506 induces weak and slow phosphorylation of IkappaBalpha at Ser-32. However, unlike IL-1-induced degradation, IKK-1 and IKK-2 were not activated significantly nor was FK506-induced IkappaBalpha degradation dependent on the N-terminal ubiquitination sites (Lys-21 and Lys-22). These results therefore indicate that FK506 and IL-1 utilize similar but distinct mechanisms to induce the phosphorylation and degradation of IkappaBalpha.