The atria and ventricles of the heart have distinct development, structure, and physiology. However, only a few of the genes that underlie the differences between these tissues are known. We used a murine cardiac cDNA microarray to identify genes differentially expressed in the atria and ventricles. The reliability of these findings is supported by highly concordant repetition of hybridization, recognition of previously known atrial and ventricular isoforms of contractile proteins, and confirmation of results by quantitative PCR and in situ hybridization. We examined the most differentially regulated genes for evolutionarily conserved noncoding sequences and found that atrial-expressed genes have more predicted myocyte enhancer factor-2 (MEF2) binding sites than ventricle-predominant genes. We confirmed that messages for MEF2 family members are more abundant in the atria, as are their protein products. Moreover, the activity of a transgenic reporter construct for MEF2 activity is preferentially upregulated in the atria in response to hypertrophic stimuli. This study provides a greater understanding of the molecular differences between atria and ventricles and establishes the framework for an anatomically detailed evaluation of cardiac transcriptional regulation.