The sequence of the 5'-flanking region of the rat Mrp3 gene was determined up to 2723 bp upstream of the translation start site. Regulatory regions crucial for Mrp3 promoter activity were characterized between -157 and -106 bp in hepatoma cells. Within this sequence we identified putative binding sites for C/EBP and Sp1. EMSA and supershift assays demonstrated specific binding of Sp1, Sp3, C/EBPalpha, beta, and delta. In Drosophila SL2 cells, both Sp1 and Sp3 transactivated the Mrp3 minimal promoter (pWT-157). Structural and functional analysis demonstrated that binding sites for C/EBPs, Sp1 and Sp3 were essential for transcription of the rat Mrp3 gene in Mrp3-expressing cells (including: H4IIE, H4IIE C3, BRL 3A, Clone 9, and RAT 2). Cotransfection assays demonstrated that C/EBP transcription factors modulated the basal and tissue specific activity of the Mrp3 gene promoter by recognition of the C/EBP (-157/-140) element and through functional cooperation with factors interacting with the Sp1 (3) and Sp1 (4) (-140/-106) cis-acting elements. In this study, we found C/EBPs and Sp1/Sp3 cooperatively regulated the promoter activity of rat Mrp3 gene through proximal (-157/-106) region. It suggested another fine-tune regulation mechanism may involve in Mrp3 gene expression.