Enhancer of split [E(spl)] refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway. The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)). The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D). This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein. Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed. In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level. In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype. This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins. Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl). In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation. However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity. Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed. These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl).