Cell-cell interactions mediated by the Notch receptor play an essential role in the development of the Drosophila adult peripheral nervous system (PNS). Transcriptional activation of multiple genes of the Enhancer of split Complex [E(spl)-C] is a key intracellular response to Notch receptor activity. Here we report that most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3′ untranslated regions (3′ UTRs). We present three lines of evidence that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. First, K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species. Second, the wild-type m8 3′ UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences. Finally, m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, we find that K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, we present sequence analyses that implicate genes of the iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation.