Notch signaling pathway genes are required for nephrogenesis, raising the possibility that Notch effector Hairy-related genes should also control nephron formation. We performed in situ hybridization of Hairy transcription factors with segment-specific lectins and/or antibodies during early nephrogenesis to identify their possible roles in segment identity of the nephron. We found that among all of Notch downstream Hairy genes, only Hes1, Hes5, Hey1, and HeyL were expressed in a segment-specific manner in early nephrons and their expression pattern changed dynamically during metanephric development. Based on these patterns of expression, it was possible to propose a pairwise association of specific ligand and receptor and to suggest that the effector of this association is one of the Hairy transcription factors. We found that Hes5 is specifically expressed in the anlage of the loop of Henle, suggesting that it might be involved in the determination of its cell identity. We also examined the morphological appearance of kidneys from mice where the Hes1 or Hes5 genes were deleted and found that at least at the gross morphological level, there was little difference from wild-type kidneys. Because Hairy genes associate with other transcription factors to exert their effect, it is necessary to examine a more complete array of genetic deletions before a conclusion can be reached regarding their role in kidney development. These studies provide the basis for the future development of strategies to examine the role of individual effector molecules in the determination of the differentiation pattern of the nephron.