The upstream glucokinase (GK) promoter is expressed specifically in several different neural/neuroendocrine (NE) cell types, including the pancreatic beta-cell and pituitary corticotrope. Previously, a mutational and evolutionary analysis of this promoter identified two identical 9-bp motifs (TGGTCACCA) termed Pal-1 and Pal-2 that are essential for high level expression in HIT M2.2.2 cells, an insulinoma cell line. Here we show that these motifs are also necessary for efficient expression in AtT-20 cells, a corticotrope-derived cell line, and that proteins from both NE and non-NE cells bind to the Pal motifs, although the DNA-protein complexes differ by cell type. Complexes formed using nuclear extracts from NE cells contained an extra NE cell-specific band and differed in the relative abundance of two other bands when compared with non-NE cells, UV laser cross-linking experiments further supported the cell-specific binding of two proteins, 110 and 150 kDa in size, to these motifs. The presence or absence of the NE-specific band correlates with transcription of GK promoter fusion gene constructs, suggesting a key role for this protein in determining the cell-specific expression of GK. The Pal motifs themselves do not function as enhancers but seem to be essential components of a larger transcriptional regulatory domain that is active only in certain NE cells. Together, these studies suggest that the NE cell-specific expression of the upstream GK promoter involves the formation of a distinct protein complex on the two Pal motifs.