The basic helix-loop-helix tal-1 gene plays a key role in hematopoiesis, and its expression is tightly controlled through alternative promoters and complex interactions of cis-acting regulatory elements. tal-1 is not expressed in normal T cells, but its transcription is constitutive in a large proportion of human T cell leukemias. We have previously described a downstream initiation of tal-1 transcription specifically associated with a subset of T cell leukemias that leads to the production of NH(2)-truncated TAL-1 proteins. In this study, we characterize the human promoter (promoter IV), embedded within a GC-rich region in exon IV, responsible for this transcriptional activity. The restriction of promoter IV usage is assured by a novel silencer element in the 3'-untranslated region of the human gene that represses its activity in erythroid but not in T cells. The silencer activity is mediated through binding of a tissue-specific nuclear factor to a novel protein recognition motif (designated tal-RE) in the silencer. Mutation of a single residue within the tal-RE abolishes both specific protein binding and silencing activity. Altogether, our results demonstrate that the tal-1 promoter IV is actively repressed in cells of the erythro-megakaryocytic lineage and that this repression is released in leukemic T cells, resulting in the expression of the tal-1 truncated transcript.