The E complex of the major histocompatibility complex (MHC) can prevent the spontaneous development of diabetes in nonobese diabetic (NOD) mice transgenic for the Ea gene. None of three promoter-mutated Ea constructs with Ea expression directed to different subsets of immunocompetent cells exerts full protection in NOD mice. The promoter-mutated constructs are all capable of mediating intrathymic elimination of I-E-restricted T cells. Thus, thymic negative selection is not responsible for the protective effect but a more complex effect is likely. Here we show that combinations of two or three different mutated Ea constructs do not protect against intra-islet insulitis either. We also show that spleen cells from protected animals are sufficient to protect NOD mice in adoptive transfer experiments. The only detectable expression defects in splenic cells or cells influencing the repertoire of splenic cells are in the B-cell compartment. Furthermore, in three construct combinations, the differences to wild-type expression are extremely small. Thus, we conclude that even minute disturbances of the E expression pattern might reduce the protection of NOD mice from insulitis.