GATA-4 is a key regulator of a poorly understood cardiac morphogenetic program. We used genomic regions of the GATA-4 gene to target GFP expression to the developing heart of living fish. In these fish, GFP-expressing cells in the lateral plate mesoderm form two tubes that migrate ventrally to fuse into a linear heart tube. In addition, we find that a 14.8-kb fragment upstream of the transcription initiation site targets expression to both chambers and the valves of the heart. Truncation of 7 kb of the distal sequences eliminates expression in the atrium and the atrioventricular valve while expression is retained in the ventricle and the bulboventricular valve. Within this 7-kb distal regulatory region, we delineated a 1300-bp region with a cluster of consensus binding sites for T-box transcription factors. Mutation of these sequences significantly reduces reporter gene expression in the heart. This provides the first evidence that T-box factors function by directly regulating GATA-4 expression. Thus, GATA-4 regulatory elements control gene expression differentially along the rostro-caudal axis, and T-box binding elements in the GATA-4 promoter contribute to heart-specific expression.