We investigated chamber-specific gene expression by isolating a 2.2-kb polymerase chain reaction product containing the 5′-flanking region of the zebrafish ventricular myosin heavy-chain gene (vmhc). Promoter analysis revealed that the fragment, consisting of nucleotides from −301 to +26, is sufficient for vmhc promoter activity. Among several putative cis-acting elements in the region, a GATA-binding site was identified to be crucial for the activity of the promoter, as evidenced by the complete abolishment of promoter activity by a single nucleotide substitution of GATA-binding site (−287, C→T). Knockdown of GATA-binding proteins 4 and 6 (GATA4 and -6) by their antisense morpholino oligonucleotides resulted in reduced green fluorescent protein (GFP) reporter gene and endogenous vmhc expression. These findings suggest that GATA4 and -6 play a key role in the regulation of vmhc expression in the ventricle. In addition, the vmhc promoter and the transgenic zebrafish (vmhc:gfp) are useful tools to study the formation and function of the ventricle. Developmental Dynamics 238:1574–1581, 2009. © 2009 Wiley-Liss, Inc.