The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Here, we demonstrate that the slow/cardiac-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage- restricted, zinc finger transcription factor, GATA-4 and that GATA-4 mRNA and protein is expressed in cardiac myocytes. In addition, GATA-4 binding sites were identified in several previously characterized cardiac-specific transcriptional regulatory elements. The cTnC GATA-4 binding site is required for transcriptional enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non- cardiac muscle cells such as NIH 3T3 cells. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.