The purpose of this study was to determine the rate of oxygen consumption in mouse aortic endothelial cells (MAECs) and to determine the effect of a variety of inhibitors and stimulators of oxygen consumption measured by electron paramagnetic resonance (EPR) spectroscopy utilizing a new particulate oximetry probe. We have previously demonstrated that the octa-n-butoxy derivative of naphthalocyanine neutral radical (LiNc-BuO) enables accurate, precise, and reproducible measurements of pO(2) in cellular suspensions. In the current study, we carried out measurements to provide an accurate determination of pO(2) in small volume with less number of cells (20,000 cells) that has not been possible with other techniques. To establish the reliability of this method, agents such as menadione, lipopolysaccharide (LPS), potassium cyanide, rotenone, and diphenyleneiodonium chloride (DPI) were used to modulate the oxygen consumption rate in the cells. We observed an increase in oxygen consumption by the cells upon treatment with menadione and LPS, whereas treatment with cyanide, rotenone, and DPI inhibited oxygen consumption. This study clearly demonstrated the utilization of EPR spectrometry with LiNc-BuO probe for determination of oxygen concentration in cultured cells.