Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of inflammatory leukotrienes. 5-LO gene expression is mainly restricted to B cells and cells of myeloid origin. It is known that basal 5-lipoxygenase promoter activity is regulated by DNA methylation. In this study we investigated the impact of the DNA methylation status of the 5-LO promoter on its activity and the role of methyl DNA binding proteins (MBDs) in transcriptional silencing of the 5-LO promoter. Using ChIP assays, we found that the methyl-DNA binding proteins MBD1, MBD2 and MeCP2 bind to the methylated 5-LO core promoter in U937 cells. Knock down of each of the MBDs upregulates 5-LO mRNA expression in U937 cells indicating that these proteins are involved in silencing of the 5-LO gene. In reporter gene assays with in vitro methylated 5-LO promoter constructs, the extent of 5-LO promoter methylation inversely correlated with its activity. Furthermore, we found that MBD1 overexpression repressed 5-LO promoter activity when the CpG sites at the Sp1 binding site close to the transcriptional start site (GC4) were methylated. Gel shift data indicate that recruitment of Sp1 to this binding site is prevented by methylation.