Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.