Rett syndrome (RTT) is an X-linked dominant disabling neurodevelopmental disorder caused by loss of function mutations in the MECP2 gene, located at Xq28, which encodes a multifunctional protein. MECP2 expression is regulated in a developmental stage and cell-type-specific manner. The need for tightly controlled MeCP2 levels in brain is strongly suggested by neurologically abnormal phenotypes of mouse models with mild overexpression and by mental retardation in human males with MECP2 duplication. We set out to identify long-range cis-regulatory sequences that differentially regulate MECP2 transcription and, when mutated, may contribute to the pathogenesis of RTT, autism or X-linked mental retardation. By inter-species sequence comparisons, we detected 27 highly conserved non-coding DNA sequences within a 210 kb region covering MECP2. We functionally confirmed four enhancer and two silencer elements by performing luciferase reporter assays in four different human cell lines. The transcription factor binding capability of the identified regulatory elements was tested by gel shift assays. To locate the human MECP2 core promoter, we dissected the promoter region by reporter assays with deletion constructs. We then used chromosome conformation capture methods to document long-range interactions of three enhancers and two silencers with the MECP2 promoter. Acting over distances of up to 130 kb, these elements may influence chromatin configurations and regulate MECP2 transcription. Our study has defined the "MECP2 functional expression module" and identified enhancer and silencer elements that are likely to be responsible for the tissue-specific, developmental stage-specific or splice-variant-specific control of MeCP2 protein expression.