The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between -187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA-1 rather than the presence of trans-dominant negative regulators.