In the mature pancreas, the homeodomain transcription factor Nkx6.1 is uniquely restricted to beta-cells. Nkx6.1 also is expressed in developing beta-cells and plays an essential role in their differentiation. Among cell lines, both beta- and alpha-cell lines express nkx6.1 mRNA; but no protein can be detected in the alpha-cell lines, suggesting that post-transcriptional regulation contributes to the restriction of Nkx6.1 to beta-cells. To investigate the regulator of Nkx6.1 expression, we outlined the structure of the mouse nkx6.1 gene, and we identified regions that direct cell type-specific expression. The nkx6.1 gene has a long 5'-untranslated region (5'-UTR) downstream of a cluster of transcription start sites. nkx6.1 gene sequences from -5.6 to +1.0 kilobase pairs have specific promoter activity in beta-cell lines but not in NIH3T3 cells. This activity is dependent on sequences located at about -800 base pairs and on the 5'-UTR. Electrophoretic mobility shift assays demonstrate that homeodomain transcription factors PDX1 and Nkx2.2 can bind to the sequence element located at -800 base pairs. In addition, dicistronic assays establish that the 5'-UTR region functions as a potent internal ribosomal entry site, providing cell type-specific regulation of translation. These data demonstrate that complex regulation of both Nkx6.1 transcription and translation provides the specificity of expression required during pancreas development.