Mitochondria represent a major source of reactive oxygen species (ROS) particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins is to decrease mitochondrial membrane potential and, thereby, protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by uncoupling proteins (UCPs). Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance (EPR) spectroscopy, was approximately 4-fold greater during state 4 compared to state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, in spite of uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-[trifluoromethoxyl]-phenyl-hydrazone (FCCP). In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria, but that uncoupling by UCP1 or UCP2 does not prevent acute substrate driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.