The Maf family of the transcription factors plays a pivotal role in controlling development and cellular differentiation. To clarify the molecular mechanisms controlling mafB expression, a genomic clone of the mouse mafB gene was isolated and analyzed. RNase protection analysis determined the transcription initiation site at 389 bp upstream from the translation initiation site. The 3' end of the gene is located at 946 bp downstream from the termination codon. The gene lacks intron structure. Sequence analysis showed a TATA-like sequence (5'-GATAAAA-3') and an inverted CCAAT-box (5'-ATTGG-3') in the promoter region. Upstream of these sequences, there are several potential regulatory elements, including two GC-boxes (5'-GGGCGG-3'), and a palindromic sequence (5'-GTCAGCTGAC-3') which contains two Maf recognition elements (MARE, 5'-GCTGAC-3') and an E-box (5'-CAGCTG-3'). Transient transfection analysis with the 5'-flanking region of the mafB gene demonstrated that these elements are important for mafB gene expression. In addition, cotransfection analysis indicated that the MyoD activates the mouse mafB promoter and the gene is positively auto-regulated by its own product.