ABSTRACT: BACKGROUND: GATA-2 is a transcription factor required for hematopoietic stem cell survival as well as for neuronal development in vertebrates. It has been shown that specific expression of GATA-2 in blood progenitor cells requires distal cis-acting regulatory elements. Identification and characterization of these elements should help elucidating transcription regulatory mechanisms of GATA-2 expression in hematopoietic lineage. RESULTS: By pair-wise alignments of the zebrafish genomic sequences flanking GATA-2 to orthologous regions of fugu, mouse, rat and human genomes, we identified three highly conserved non-coding sequences in the genomic region flanking GATA-2, two upstream of GATA-2 and another downstream. Using both transposon and bacterial artificial chromosome mediated germline transgenic zebrafish analyses, one of the sequences was established as necessary and sufficient to direct hematopoietic GFP expression in a manner that recapitulates that of GATA-2. In addition, we demonstrated that this element has enhancer activity in mammalian myeloid leukemia cell lines, thus validating its functional conservation among vertebrate species. Further analysis of potential transcription factor binding sites suggested that integrity of the putative HOXA3 and LMO2 sites is required for regulating GATA-2/GFP hematopoietic expression. CONCLUSION: Regulation of GATA-2 expression in hematopoietic cells is likely conserved among vertebrate animals. The integrated approach described here, drawing on embryological, transgenesis and computational methods, should be generally applicable to analyze tissue-specific gene regulation involving distal DNA cis-acting elements.