&NA;Endocardial endothelium and endothelium of coronary vessels produce NO. Histochemical methods have suggested that coronary arterial endothelial cells contain more endothelial constitutive NO synthase (ecNOS) than does coronary venous endothelium. We have further investigated the distribution of ecNOS in cardiac endothelium using immunofluorescence and en face confocal microscopy of rat heart. In endocardial endothelium, confocal microscopy revealed distinct ecNOS labeling of peripheral cell borders, cytoplasmic labeling, and labeling of the Golgi complexes. Labeling of the cell borders and of the Golgi complexes was confirmed by double staining for ecNOS and for platelet and endothelial cell adhesion molecule or Golgi 58k protein, respectively. Cytoplasmic labeling was strongest in coronary arterial endothelium. The size of the ecNOS‐labeled Golgi complexes decreased from coronary arterial endothelial cells (8.63 +/‐ 0.39 [micro sign]m2, mean +/‐ SE of 5 rats) to endocardial endothelium (7.07 +/‐ 0.61 [micro sign]m2) and to coronary venous endothelium (3.65 +/‐ 0.20 [micro sign]m2). In addition, pixel intensity of ecNOS labeling was higher in arterial endothelial cells than in venous endothelial cells. Endothelium of myocardial capillaries also contained small ecNOS‐labeled Golgi complexes. No correlation was observed between endothelial cell surface area and Golgi complex size. Caveolin‐1 labeling was strongest in capillaries and did not coincide completely with ecNOS labeling in endocardial and venous endothelium. These results suggest that endocardial and coronary arterial endothelium in the rat have a higher synthetic activity and might express more ecNOS than is expressed by cardiac venous and capillary endothelium. The observed heterogeneity in ecNOS distribution might be related to the specific mechanochemical environment and function of each endothelial compartment. (Circ Res. 1998;82:195‐203.)