Background: Investigations involving biopsies of human cardiac tissue often assume that myocardial samples from a specific location are representative of the entire heart. Hypothesis: There are significant regional differences in gene expression in the heart. Methods: We used two models. In the first model, seven whole human hearts were cut in 1-cm slices from apex to base and 11 distinct regions were sampled. Full thickness left ventricular tissue was further subdivided equally into an inner, outer, and middle region. In the second model, hearts were removed from adult Sprague-Dawley rats and were divided into 4 regions. Using species-specific quantitative reverse transcriptase-polymerase chain reaction, we measured transcript levels of myosin heavy chain β (MHC-β), glucose transporter 1 (GLUT 1), and atrial natriuretic factor (ANF) in tissue samples from both models. Results: In human heart, there were significant differences in transcript levels between regions. The following patterns could be recognized among the seven hearts. ANF expression was highest in the subendocardial region. MHC-β and GLUT 1 transcript levels were higher in the right ventricle than the left ventricle. As expected, ANF transcript levels were highest in the atria, where MHC-β and GLUT 1 expression was low. Analogous to the human studies, MHC-β and GLUT 1 transcript levels were low in rat atria as compared to ventricles. In rat heart, MHC-β expression was higher in the left ventricle than the right ventricle while GLUT 1 expression was not significantly different between ventricles. Conclusion: Despite the large variability in transcript levels among different regions in human hearts, certain patterns in gene expression emerged suggesting that different anatomical regions of the heart also differ in respect to gene expression.