Vascular generation of nitric oxide (NO) plays an important role in the regulation of blood flow, and is counterbalanced by the formation of radical oxygen species (ROS). Thus, an imbalance in vascular NO and ROS production contributes to endothelial dysfunction, which is associated with cardiovascular disease. Here we review the main and commonly used methods that have been applied to determine vascular NO and ROS. Only NO microsensors, electron spin resonance (ESR), and NO sensitive fluorescent probes allow real time measurement of NO levels in cell suspensions, cell cultures, and isolated vascular segments in vitro, and of these techniques only NO microsensors have so far been developed for real time detection of NO in vivo. ROS formation has been detected in cell cultures by lucigeninenhanced fluorescence, fluorescent probes e.g. 2',7'-dichlorodihydrofluorescein and dihydroethidium, by use of ESR, and also by electrochemical detection. The limitations of the electrochemical microsensors include interference from other substances, physical force on the sensors, and influence of temperature and changes in pH that may lead to artifactual changes in current. Therefore, specificity testing and calibration are pivotal, and in case of ROS, application of more than one technique is recommendable. Technical advances have allowed simultaneous detection of both NO and superoxide anion by use of electrochemical microsensors. Miniaturizing the sensors may also allow incorporation of those in lab-ona- chip and may lead to real time measurements of NO and ROS in the coronary circulation in situ, and hence provide direct evaluation of endothelial and vascular function in cardiovascular disease.