S100A9, also referred to as MRP14, is a calcium-binding protein whose expression is tightly regulated during differentiation of myeloid cells. The present study was performed to study the cell type- and differentiation-specific transcriptional regulation of the S100A9 gene. Analysis of the S100A9 promoter in MonoMac-6 cells revealed evidence for a novel regulatory region from position -400 to -374 bp, termed myeloid-related protein regulatory element (MRE). MRE deletion resulted in a 5.2-fold reduction of promoter activity. By electrophoretic mobility shift analysis two nuclear complexes binding to this region were identified and referred to as MRE-binding complex A (MbcA) and MRE-binding complex B (MbcB). By mutagenesis the MRE-binding motif could be narrowed to a 12-bp region. The relevance of MRE is deduced from the observations that the formation of either MRE-binding complex A or MRE-binding complex B strongly correlated with S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner. Moreover, DNA affinity chromatography and Western blot studies indicate that a Kruppel-related zinc finger protein and the transcriptional intermediary factor 1beta (TIF1beta) are involved in an MRE-binding complex, thereby regulating the S100A9 gene expression.