Processing of the NF-kappaB2 precursor protein p100 to generate p52 is an important step of NF-kappaB regulation. This proteolytic event is tightly regulated by sequences located at the C-terminal portion of p100. Constitutive processing of p100 occurs in certain lymphoma cells due to the loss of its C-terminal regulatory domain, although the underlying mechanisms remain unknown. We show here that the constitutive processing of C-terminal truncation mutants of p100 is associated with their active nuclear translocation. Deletion of the C-terminal death domain of p100 triggers a low, but significant, level of nuclear translocation and processing. Disruption of the ankyrin-repeat domain of p100 further enhances its nuclear shuttling activity, which is again associated with elevated level of processing. More importantly, mutation of the nuclear localization signal (NLS) of p100 abolishes its processing, and this defect can be rescued by fusion of a heterologous NLS to the amino- or carboxyl-terminus of the p100 mutant. These results suggest that nuclear shuttling is a mechanism regulating the processing of NF-kappaB2/p100.