Members of the GATA family of zinc finger transcription factors play important roles in the development of several mesodermally derived cell lineages. In the studies described in this report, we have isolated and functionally characterized the murine GATA-6 cDNA and protein and defined the temporal and spatial patterns of GATA-6 gene expression during mammalian development. The GATA-6 and -4 proteins share high-level amino acid sequence identity over a proline-rich region at the amino terminus of the protein that is not conserved in other GATA family members. GATA-6 binds to a functionally important nuclear protein binding site within the cardiac-specific cardiac troponin C (cTnC) transcriptional enhancer. Moreover, the cTnC promoter enhancer can be transactivated by overexpression of GATA-6 in noncardiac muscle cells. During early murine embryonic development, the patterns of GATA-6 and -4 gene expression are similar, with expression of GATA-6 restricted to the precardiac mesoderm, the embryonic heart tube, and the primitive gut. However, coincident with the onset of vasculogenesis and development of the respiratory and urogenital tracts, only the GATA-6 gene is expressed in arterial smooth muscle cells, the developing bronchi, and the urogenital ridge and bladder. These data are consistent with a model in which GATA-6 functions in concert with GATA-4 to direct tissue-specific gene expression during formation of the mammalian heart and gastrointestinal tract, but performs a unique function in programming lineage-restricted gene expression in the arterial system, the bladder, and the embryonic lung.