MafA and Pdxl represent critical transcriptional regulators required for the maintenance of pancreatic islet β-cell function. The in vivo β-cell-enriched expression pattern of these genes is principally directed by islet transcription factors binding within conserved Region 3 (base pairs (bp) - 8118/-7750) oiMafA and Area II (bp -2153/-1923) of the Pdxl gene. Comprehensive mutational analysis of conserved MafA Region 3 revealed two new β-cell line-specific cw-activation elements, termed Site 4 (bp -7997 to -7988) and Site 12 (bp -7835 to -7826). Gel mobility and antibody super-shift analysis identified Pdxl as the Site 4 binding factor, while an 80 - 88 kilodalton (kDa) β-cell line-enriched protein complex bound Site 12 and similar aligned nucleotides within Pdxl Area II. The 80-88 kDa activator was also found in adult mouse islet extract. Strikingly, the molecular weight, DNA binding, and antibody recognition properties of this activator were unique when compared with all other key islet transcription factors tested, including Proxl (83 kDa), Hnflα (67 kDa), FoxA2 (48 kDa), MafA (46 kDa), Isll (44 kDa), Pdxl (42 kDa), and Nkx2.2 (30 kDa). Collectively, these data define an apparently novel MafA Region 3 and Pdxl Area II activator contributing to expression in β-cells. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.