GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. The transcriptional regulation of GATA-3, however, remains elusive. In this study, we report the identification of a regulatory region located within the first intron of the murine GATA-3 gene. The intronic regulatory region contains both a positive and a negative cis-acting element but, as a whole, serves as a potent T cell-specific enhancer and is essential for the promoter activity in vitro. By using yeast one-hybrid screening, we discovered that fetal liver zinc finger protein 1 (Fliz1) could bind specifically to the negative cis-acting element, the sequence of which is conserved between the mouse and human GATA-3 genes. More importantly, overexpression of Fliz1 repressed the expression of GATA-3 in vivo and in vitro. Our data suggest that the expression of GATA-3 might be partly regulated by the intronic regulatory region and Fliz1 in a developmental stage-specific fashion.