In vertebrates, heart development is a multistep process that starts with formation and patterning of the primitive heart tube and is followed by complex morphological events to give rise to the mature four-chambered heart. These various stages are characterized by distinct patterns of gene expression. Although chamber specificity and developmental regulation can be demonstrated in transgenic mice using short promoter fragments, the mechanism underlying spatial and temporal specificity within the heart remains largely unclear. Combinatorial interaction between a limited number of cardiac-specific and ubiquitous transcription factors may account for the diverse genetic inputs required to generate the complex transcriptional patterns that characterize the developing myocardium. We have used the cardiac atrial natriuretic peptide (ANP) promoter to test this hypothesis. The ANP gene is transcribed in a spatialand temporal-specific manner in the heart, and a 500 bp promoter fragment is sufficient to recapitulate both chamber and developmental specificity. This promoter is composed of three modules, a ''basal'' cardiac promoter that is essential for transcription in embryonic and postnatal atrial and ventricular myocytes and two other independent modules that behave as chamber-specific enhancers. The basal cardiac promoter is the target of two cardiac-specific transcription factors, the zinc finger GATA-4 protein and the Nkx2-5 homeodomain, which bind to contiguous elements within this region. At low concentrations-a situation that likely occurs during the very first stages of cardiac cell fate determination-the two proteins synergistically activate transcription from the ANP promoter. This functional synergy requires physical interaction between the GATA-4 protein and an extended C-terminal homeodomain on Nkx2-5. This interaction, which unmasks an activation domain present just N-terminal of the homeodomain, is specific for GATA-4 and -5, but is not observed with the other cardiac GATA factor, GATA-6. Optimal synergy requires binding of both proteins to their cognate sites, although modest synergy also could be observed on heterologous promoters containing only multimerized Nkx binding sites, suggesting that Nkx2-5 is able to recruit GATA-4 into a transcriptionally active complex. The GATA/Nkx interac-tion, which appears to have been evolutionary conserved in nematode, fly, and mammals, provides a paradigm for analyzing transcription factor interaction during organogenesis. The data are also discussed in the context of our present knowledge of the roles of GATA and NK2 proteins in cardiac development. Dev.