Myelin basic protein (MBP) is required for normal myelin compaction and is implicated in both experimental and human demyelinating diseases. In this study, as an initial step in defining the regulatory network controllingMBPtranscription, we located and characterized the function of evolutionarily conserved regulatory sequences. Long-range human-mouse sequence comparison revealed over 1 kb of conserved noncodingMBP5′ flanking sequence distributed into four widely spaced modules ranging from 0.1 to 0.4 kb. We demonstrate first that a controlled strategy of transgenesis provides an effective means to assign and compare qualitative and quantitativein vivoregulatory programs. Using this strategy, single-copy reporter constructs, designed to evaluate the regulatory significance of modular and intermodular sequences, were introduced by homologous recombination into the mousehprt(hypoxanthine-guanine phosphoribosyltransferase) locus. The proximal modules M1 and M2 confer comparatively low-level oligodendrocyte expression primarily limited to early postnatal development, whereas the upstream M3 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. Mutation of putative Nkx6.2/Gtx sites within M3, although not eliminating oligodendrocyte targeting, significantly decreases transgene expression levels. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. In addition, when isolated from surroundingMBPsequences, M3 confers transient expression to Schwann cells elaborating myelin. These observations define thein vivoregulatory roles played by conserved noncodingMBPsequences and lead to a combinatorial model in which different regulatory modules are engaged during primary myelination, myelin maintenance, and remyelination.