Three major inhibitors of the NF-κB/Rel family of transcription factors, IκBα, IκBβ, and IκBε, have been described. To examine the in vivo role of the most recently discovered member of the IκB family, IκBε, we generated a null allele of the murine IκBε gene by replacement of all coding sequences with nlslacZ. Unlike IκBα nullizygous mice, mice lacking IκBε are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of β-galactosidase expression pattern revealed that IκBε is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44− CD25+ T cell subspecies. The IκBε-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-κB/Rel-binding activity is probably due to compensatory mechanisms involving IκBα and IκBβ, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1α, IL-1β, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IκBε-null macrophages in contrast to GM-CSF, G-CSF, and IFN-γ, which remain undetectable.