The mammalian heart begins contracting at the linear tube stage during embryogenesis and continuously pumps, nonstop, throughout the entire lifetime of the animal. Therefore, the cardiac energy metabolizing pathways must be properly established and efficiently functioning. While the biochemistry of these pathways is well defined, limited information regarding the regulation of cardiac metabolic genes is available. Previously, we reported that 1.9 kilobase pairs of murine adenylosuccinate synthetase 1 gene (Adss1) 5'-flanking DNA directs high levels of reporter expression to the adult transgenic heart. In this report, we define the 1.9-kilobase pair fragment as a cardiac-specific enhancer that controls correct spatiotemporal expression of a reporter similar to the endogenous Adss1 gene. A 700-base pair fragment within this region activates a heterologous promoter specifically in adult transgenic hearts. Proteins present in a cardiac nuclear extract interact with potential transcription factor binding sites of this region and these cis-acting sites play important regulatory roles in the cardiac expression of this reporter. Finally, we report that several different cardiac transcription factors trans-activate the 1.9HSCAT construct through these sites and that combinations result in enhanced reporter expression. Adss1 appears to be one of the first target genes identified for the bHLH factors Hand1 and Hand2.