Homeodomain transcription factor Nkx2.2 is required for the final differentiation of the beta-cells in the pancreas and for the production of insulin. Nkx2.2 is expressed in islet cell precursors during pancreatic development and persists in a subset of mature islet cells including all beta-cells. To understand the mechanisms regulating the expression of Nkx2.2 in these different cell populations, we outlined the structure of the mouse nkx2.2 gene and identified regions that direct cell type-specific expression. The nkx2.2 gene has two noncoding alternative first exons (exons 1a and 1b). In transgenic mice, sequences upstream from exon 1a directed expression predominantly in mature islet cells. Within this exon 1a promoter, cooperative interactions between HNF3 and basic helix-loop-helix factors neurogenin-3 or NeuroD1 binding to adjacent sites played key roles in its islet cell-specific expression. In contrast, sequences upstream from exon 1b restricted expression specifically to islet cell precursors. These studies reveal distinct mechanisms for directing the expression of a key differentiation factor in precursors versus mature islet cells.