The basic helix-loop-helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in β-cells and either absent or expressed below the limit of lacZ detection in mature α-, δ-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1 with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic δ-cell line, resulted in potent repression of somatostatin concomitant with induction of the β-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in β- but not δ-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously.