The cardiac sarcolemmal Na⁺/Ca²⁺ exchanger plays a primary role in Ca²⁺ efflux and is important in regulating intracellular Ca²⁺ and beat-to-beat contractility. Of the three Na⁺/Ca²⁺ exchanger genes cloned (NCX1, NCX2, and NCX3), only NCX1 is expressed in cardiac myocytes. NCX1 has alternative promoters for heart, kidney, and brain tissue-specific transcripts. Analysis of the cardiac NCX1 promoter (at 336 bp) identified a cardiac-specific minimum promoter (at 137) and two GATA sites (at 75 and 145). In this study, gel shift and supershift analyses identified GATA-4 in primary neonatal cardiac myocytes. Site-directed mutagenesis of the GATA-4 site at 75 abolishes binding and reduces activity of the minimum and full-length promoters by >90 and ~60%, respectively. Mutation of the GATA site at 145 reduces activity of the full-length promoter by ~30%. Mutation of an E-box at 175 does not alter promoter activity.