The myelin sheath, a structure exclusive to vertebrates, is composed of lipid-rich membranous processes produced by specialized classes of glia, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Axons are ensheathed by compact myelin internodes that facilitate the fast propagation of action potentials along their length via myelin free nodes of Ranvier. Myelin basic protein (Mbp) is essential for myelin compaction in the CNS. Although the composition and function of myelin is well established, less is known about the factors that regulate myelination or the molecules that stabilize the myelin sheath. I have taken advantage of the zebrafish system to generate stable transgenic zebrafish lines that label oligodendrocytes with green fluorescent protein (GFP) under the control of Mbp regulatory regions. I generated zebrafish transgenic lines using previously characterized mouse Mbp regulatory modules (Farhadi et al., 2003), alone or in combination, and GFP labeling was confirmed to localize to oligodendrocytes. The effective mouse regulatory modules shared a Sox10 HMG-box consensus binding site and two identical Olig1/Olig2 E-box binding sites in reverse orientation. The same three transcription factor recognition sequences were found within defined regions upstream of the human, chicken, puffer fish and zebrafish mbp genes. The results suggest that a triplet transcription factor binding site motif, comprised of conserved Sox10 and Olig1/2 recognition sequences, is sufficient to activate mbp expression in myelinating oligodendrocytes. To investigate whether olig1 or olig2 is capable of regulating mbp transcription and myelination, I depleted olig1 and olig2 by antisense morpholinos. The data show that olig2 depletion caused reduced mbp expression and loss of myelin in the spinal cord, while mbp expression and myelin in the hindbrain remained. Depletion of olig1 caused an increase in the number of oligodendrocytes expressing myelin-specific genes and excess myelin formation was observed. The data suggest that Olig2 is essential for mbp transcription and myelination in the spinal cord, while the absence of Olig1 caused more oligodendrocytes to differentiate into myelinating glia. Therefore, I propose that Olig1 normally functions to regulate the differentiation of oligodendrocyte precursor cells to mature oligodendrocytes.