Oxygen saturation of small arteries and veins (20-500 micron) was determined microspectrophotometrically in the hearts of 12 pentobarbital-anesthetized open-chest dogs. Hearts were removed, quick frozen in liquid propane, and O2 saturation was determined in blood vessels on a regional basis between and within ventricular walls. No significant differences existed in arterial O2 saturation between right, left, and septal walls or regionally within any wall by depth or in base-to-apex comparisons. Although there was variation in arterial saturation, it was independent of vessel size. Arteries were followed by serial section into the left ventricular wall for distances up to 7.5 mm without significant saturation change. The average venous saturations of the right, septal, and left ventricular walls were not significantly different. No regional differences in venous saturation were found within any ventricular wall in comparisons between base and apex. In the left ventricle, subepicardial venous saturation (29.8%) was significantly higher than subendocardial saturation (16.4%). In veins traced from the surface, saturation decreased with depth. Greater variability of saturation was found in small compared to large veins. The greater O2 extraction in the subendocardium may indicate a higher O2 consumption than in the subepicardium.