Notch signaling is used to specify cell types during animal development. A high level specifies one cell type, whereas a low level specifies the alternate type. The effector of Notch signaling is the Notch intracellular domain. Upon its release from the plasma membrane in response to Delta binding the Notch extracellular domain, the Notch intracellular domain combines with the transcription factor Suppressor of Hairless and promotes the expression of target genes. Using a panel of antibodies made against different extracellular and intracellular regions of Notch, we show that cell types and tissues with low levels of Notch signaling are enriched for Notch molecules detected only by the extracellular domain antibodies. This enrichment often follows enrichment for Notch molecules detected only by antibodies made against the Suppressor of Hairless binding region. Notch molecules lacking most of the intracellular domain or containing only the Suppressor of Hairless binding region are produced during development. Such molecules are known to suppress Notch signaling, possibly by taking away Delta or Suppressor of Hairless from the full-length Notch. Thus, it is possible that dominant-negative Notch molecules are produced in the normal course of tissue differentiation in Drosophila as part of an auto–down-regulation mechanism. Developmental Dynamics 235:411–426, 2006. © 2005 Wiley-Liss, Inc.