PLZF (promyelocytic leukemia zinc finger ) is a transcription factor disrupted in t(11;17)-associated acute promyelocytic leukemia which is highly expressed in undifferentiated myeloid cells. To address the tissue-specific regulation of the promoter, we isolated sequences 1.2-kb 5' to the transcriptional start site. Sequence analysis demonstrated that this region contains one TATA box and several putative transcription factor binding sites including four G/C-rich sites and one Evi-1-like site. A fragment of the promoter spanning 158-bp upstream of the transcription start site displayed relative specificity for PLZF-expressing myeloid cells. Functional promoter assays revealed that an Evi-1-like site at -140/-130 was essential for full promoter activity in every cell line tested while a G-rich site at -15/-7 was important for tissue specificity. Electrophoretic mobility shift assays showed that Evi-1 binds specifically to -140/-130 Evi-1-like site and overexpression of Evi-1 in K562 cells activated the PLZF promoter. UV cross-linking assays showed that the proximal, tissue specific element at -15/-7 bound a novel 28 kDa protein. These results indicate as with other myeloid genes, a relatively small segment of DNA can direct tissue-specific expression, but unlike other myeloid promoters, no critical PU.1 or C/EBP sites were found.