To elucidate the mechanisms underlying the tissue-restricted expression of GATA factor transcription, we have isolated and analyzed the genomic chicken GATA-3 (cGATA-3) locus. Structural analysis of the clones showed that the cGATA-3 gene consists of six exons which span more than 19 kb. Two trans-activating domains and two Zn finger domains of cGATA-3 were found to be encoded separately by exons 2/3 and 4/5, respectively, indicating that each functional domain of GATA-3 is encoded by a discrete exon. We have determined 1.7 kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites. Activities of presumptive regulatory regions of this gene were assessed by transfecting chimeric constructs into a chicken T cell line MSB-1. The results showed three features of cGATA-3 gene regulation. The basal promoter activity of the cGATA-3 gene is determined by sequences lying between -104 and -29 bp of the promoter region. The upstream region containing the GATA and CACCC elements in close proximity (-1280 to -1152) appeared to act as a negative transcriptional regulator, whereas the region -1151 to -850 acts as a positive regulator. Thus, the expression of cGATA-3 gene is under complex regulation and the mode of regulation of cGATA-3 gene expression is suggested to be different from that of GATA-1 genes.