Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.Many transcription factors have been found to be members of highly related multifactor families, and thus their specificity of action must be addressed in order to ascertain their respective functions. Consequently, it is critical to determine which factor, from an array of factors with closely related DNA-binding motifs, acts upon a particular recognition site from a variety of sites with similar sequences. Equally important is the elucidation of the means by which this discrimination is achieved; a variety of mechanisms by which related transcription factors are targeted to distinct regulatory elements have been discovered. Differences in DNA-binding properties direct the zinc finger estrogen and progesterone receptors to their appropriate targets (7,44), as is the case with the Antennapedia-related homeodomain proteins Ultrabithorax and Deforned (8,9). The related retinoic acid, thyroid hormone, and vitamin D receptors exemplify a unique solution to the problem of differential target recognition by discriminating between the spacing and orientation of closely related binding sites of these obligate dimers (27,45