The C1/RIPE3b1 (− 118/− 107 bp) binding factor regulates pancreatic-β-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse βTC-3 cell nuclear extracts was purified by DNA affinity chromatography and two-dimensional gel electrophoresis. C1/RIPE3b1 binding activity was found in the roughly 46-kDa fraction at pH 7.0 and pH 4.5, and each contained N-and C-terminal peptides to mouse MafA as determined by peptide mass mapping and tandem spectrometry. MafA was detected in the C1/RIPE3b1 binding complex by using MafA peptide-specific antisera. In addition, MafA was shown to bind within the enhancer region (− 340/− 91 bp) of the endogenous insulin gene in βTC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the β-cell-enriched component of the RIPE3b1 activator. However, reverse transcription-PCR analysis demonstrated that mouse islets express not only MafA but also other members of the large Maf family, specifically c-Maf and MafB. Furthermore, immunohistochemical studies revealed that at least MafA and MafB were present within the nuclei of islet β cells and not within pancreas acinar cells. Because MafA, MafB, and c-Maf were each capable of specifically binding to and activating insulin C1 element-mediated expression, our results suggest that all of these factors play a role in islet β-cell function.