In addition to a number of common features, cardiovascular endothelium displays structural, functional, and genetic differences according to its position in the cardiovascular tree. In the heart, endocardial and cardiac microvascular endothelia (CMVE) interact directly with surrounding cardiomyocytes, whereas the endothelium within blood vessels interacts with smooth muscle cells. In this study, we investigated whether cardiac endothelial cells were distinct from aortic endothelial (AE) cells at the transcriptional level. Using Affymetrix microarray technology and subsequent real-time PCR analyses for validation, we identified sets of genes with marked preferential expression in cultured endocardial endothelium (EE) compared with cultured AE and vice versa. Among the genes preferentially expressed in EE, some were also expressed in cultured CMVE. Immunohistochemical staining of cardiac and aortic tissue revealed that the endothelial genetic diversity observed in culture reflects, in part, a physiological diversity existing in vivo. The identification of a set of genes preferentially expressed in EE provides new insights in the functional adaptations of this endothelial subtype to its intracavitary localization and to its role in the control of ventricular performance.