The Rel/NF-kappa B family transcriptional factors plays an important role in the regulation of immune and acute phase responses. The activity of Rel/NF-kappa B complexes is regulated by their interactions with members of the I kappa B family of inhibitors. We have previously shown that the RelB/p52 heterodimer is not effectively inhibited by any of the known I kappa B molecules: I kappa B alpha, I kappa B gamma and Bcl3. Here we report that the C-terminal domain of p100 (the putative I kappa B delta) functions as a strong inhibitor of RelB/p52 transcriptional activity. In vivo interaction with I kappa B delta leads to the cytoplasmic retention and decreased DNA binding activity of RelB/p52 complexes. Thus, I kappa B delta is the only I kappa B molecule able to efficiently modulate the activity of RelB/p52 heterodimer. In Daudi cells, a 46 kD protein, probably representing the C-terminal product of the proteolytic processing of p100, remains associated with Rel/NF-kappa B complexes and might have a transient regulatory function. Our results indicate a specific role for the putative I kappa B delta and suggest a possible mechanism of how the truncation of the ankyrin domain of p100, found in a number of lymphoid neoplasias, might contribute to tumorigenesis.