We have isolated and characterized the mouse gene for NDRF (neuroD-related factor), a basic helix-loop-helix transcription factor implicated in neural development and function. The gene consists of two exons and the entire protein-coding sequence is encoded by a single downstream exon. RNA blot hybridization analysis revealed that NDRF mRNA was detectable at day 4 and increased to a maximal level at day 6 during neuronal differentiation of P19 cells. To elucidate the regulatory mechanisms of the NDRF gene expression during this process, a construct containing the genomic DNA fragment of about 3 kbp upstream of the NDRF coding region fused to a luciferase reporter gene was transfected into P19 cells, and stable transformants were pooled for assay of luciferase activities. When the stable transformants were treated with RA and aggregated to induce neuronal differentiation, the luciferase activities were induced in a temporal expression pattern similar to that of the endogenous NDRF mRNA. Further experiments using a series of deletion and mutation constructs indicated that the 376-bp sequence in the 5'-flanking region of the NDRF gene is important, and that one of the E boxes in the sequence plays a critical role in the regulated expression. Transient transfection experiments also showed that the same E box is required for the transactivation of the NDRF promoter activity by neurogenin 1. These results suggest that the NDRF gene expression is regulated by an E box-binding factor during neuronal differentiation of P19 cells.