We recently published the genomic characterization of the STAT5A and STAT5B paralogous genes that are located head to head in the 17q21 chromosome and share large regions of sequence identity. We here demonstrate by transient in vitro transfection that STAT5A and STAT5B promoters are able to direct comparable levels of transcription. The expression of basal promoters is enhanced after Sp1 up-regulation in HeLa and SL2 cells while DNA methylation associated to the recruitment of MeCP2 methyl CpG binding protein down-regulates STAT5A and B promoters by interfering with Sp1-induced transcription. In addition, cross-species sequence comparison identified a bi-directional negative cis-acting regulatory element located in the STAT5 intergenic region.