NeuroD/Beta2 is a basic helix-loop-helix (bHLH) transcription factor with important functions during development of the pancreas and the nervous system. NeuroD null mutant mice die perinatally due to diabetes caused by impaired differentiation of pancreatic endocrine cells. Additionally, null mutants display severe defects in the formation of cerebellar and hippocampal granule cells, inner ear sensory neurons, and retinal photoreceptor cells. For spatio-temporally restricted inactivation of the NeuroD gene, we generated conditional mouse mutants by flanking the NeuroD coding region with loxP sites. Homozygous NeuroDloxP mutant mice are fully viable and express normal levels of NeuroD mRNA and protein. Breeding NeuroDloxP mice to Tg(mα6-Cre)B1LFR mice that express Cre recombinase under control of the GABAA receptor α6 subunit promoter resulted in efficient inactivation of the NeuroD gene in postmigratory cerebellar granule cells and a subset of brainstem nuclei. The NeuroDloxP mouse mutant will be a valuable tool to study the developmental and adult function of NeuroD in nervous system and pancreas.genesis 42:247–252, 2005. © 2005 Wiley-Liss, Inc.