In Drosophila, multiple helix-loop-helix (HLH) factors play an essential role in neural development. Mammalian homologues of such Drosophila HLH factors have been recently characterized and provide useful information for the analysis of the mechanisms of mammalian neurogenesis. We report here the molecular characterization of a novel mouse HLH factor, designated MATH-2, that has a structural homology to the product of Drosophila proneural gene atonal. MATH-2 consists of 337 amino acid residues and contains an atonal-related basic HLH domain. However, outside of this domain, there is no significant sequence similarity between MATH-2 and Atonal. MATH-2 expression occurs by embryonic day 11.5 (E11.5), and is first detected in the wall of brain vesicles as well as in the spinal cord. It is expressed in the cortical plate and the mantle layer throughout the developing central nervous system but not in the ventricular zone. By E13.5, the expression becomes more prominent in the cortical plate of the cerebrum but decreases in the other regions. In the adult, the cerebrum produces a high level of MATH-2 RNA but other neural tissues produce only low levels. MATH-2 RNA is not detected in non-neural tissues, indicating that MATH-2 expression is specific to the nervous system. The gel mobility-shift analysis shows that MATH-2 can interact with several E-box sequences in collaboration with E47, a ubiquitously expressed HLH factor. These results raise the possibility that MATH-2 may be a trans-acting factor involved in the development and maintenance of the mammalian nervous system.