Myelin formation is an evolutionarily late acquisition that contributes to fast conduction in vertebrate axons. Continuous bidirectional signaling is required for the acquisition and maintenance of the mature nerve fiber phenotype. Myelin Basic Protein (MBP) is a representative structural component of compact myelin that is co-ordinately regulated with other myelin protein genes, primarily at the transcriptional level. In an effort to define the regulatory network controlling MBP expression, I located and functionally characterized conserved regulatory sequences in 5' non-coding sequence. First, I identified four widely-spaced segments of preferential conservation in orthologous human-mouse genomic sequences (termed modules 1-4) that range from 0.1 to 0.4 kb. In order to compare qualitative and quantitative regulatory programs mediated by modular and inter-modular sequences, a novel in vivo transgenesis system was adopted and validated. Targeted insertions of single-copy reporter constructs at a predetermined location within the hprt locus allowed for direct inter-construct regulatory program comparisons. The proximal modules M1 and M2 confer relatively low-level expression in oligodendrocytes, primarily during early postnatal development. The upstream M3 module confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. M3 also confers expression to Schwann cells but only transiently during active myelin elaboration and only when isolated from surrounding MBP sequences. These observations define the regulatory roles played by a complex network of conserved non-coding MBP sequences and lead to a combinatorial model in which specific permutations of regulatory sequences are engaged differentially in various glial cell states. This experimental system also was shown to provide sufficient quantitative resolution to reveal the regulatory functions contributed by particular modular sub-domains and individual enhancer elements. M4 regulatory activity requires simultaneous contributions from elements located in both a core targeting as well as surrounding enhancing sub-domains with Sox10 and Krox-20 M4 binding appearing to mediate targeting and enhancer function, respectively. From this investigation, the complex network of elements and transcription factors that control expression of one important myelin gene has begun to emerge. This knowledge should lead to a deeper understanding of the regulatory mechanisms controlling the overall myelination program and, ultimately, to novel therapeutic strategies effective in ameliorating the consequences of inherited or acquired demyelinating disease.