We have cloned a basic helix-loop-helix (bHLH) factor gene, Bhlhb4, from a mouse beta-cell line. Fluorescence in situ hybridization (FISH) and genetic mapping place Bhlhb4 at the telomeric end of mouse chromosome 2 (H3-H4), syntenic to human chromosome 20q13. Based on phylogenetic analysis, BHLHB4 belongs to a new subgroup of bHLH factors including at least four previously identified mouse bHLH factors: BHLHB5, MIST1, OLIG1, OLIG2, and OLIG3. In the developing nervous system, Bhlhb4 was found to mark the dimesencephalic boundary, suggesting that Bhlhb4 may have a role in diencephalic regionalization. In the pancreas, Bhlhb4 is expressed in a transient fashion that suggests a role in the pancreatic endocrine cell lineage. Transfection experiments show that BHLHB4 can repress transcriptional activation mediated through the pancreatic beta-cell specific insulin promoter enhancer RIPE3. Together, these data suggest that BHLHB4 may modulate the expression of genes required for the differentiation and/or maintenance of pancreatic and neuronal cell types.