The Drosophila hindsight gene encodes a putative transcription factor containing 14 C2H2 zinc fingers and crucial in regulating epithelial morphogenesis. Two HNT DNA binding sequences, designated as HNT site A, YGGWCCA, and HNT site B, GGATGCTG, have been identified in vitro.Biochemical and cytogenetic approaches were conducted to elucidate the molecular pathways regulated by HNT. To identify the functional domains of HNT, the DNA-binding abilities of different HNT zinc-finger clusters were analyzed by gel-shift assay. The three most C-terminal zinc fingers bound HNT site A, and the adjacent two zinc fingers bound HNT site B. To identify HNT's in vivo binding sites and target genes, polytene immunostaining by alpha-HNT was performed and 57 unique HNT binding loci were identified. The strongest band, 60C, was further narrowed to 60C4∼60C7 region, covering ∼100 kb and seven genes. Collectively, the data demonstrate a DNA binding domain and seven candidate target genes of HNT.