In biological systems, reactive oxygen species (ROS) can be generated by a variety of cellular sources, among which the NADPH oxidase enzymes (NOX) are probably the major ones. They generate superoxide radicals (O₂^•¯) by electron transfer from NADPH to molecular oxygen followed by hydrogen peroxide formation. Depending on location, concentration and type, ROS are second messengers in many signalling cascades but can also act as toxins leading to induction of many pathological conditions. To understand the physiological role of ROS, simultaneous identification and quantification of these oxidants and oxygen consumption is very important. Electron spin resonance (ESR) spectroscopy can selectively detect oxygen radicals (unpaired electron spin) and enables monitoring of oxygen content using specific spin-traps and highly sensitive oximetric labels. Monocytes express high amounts of NOX2 and generate ROS during the innate immune response; a process tightly regulated by cellular Ca²⁺ homeostasis. However, several quantitative and kinetic parameters of this process are not well understood. We used cyclic hydroxylamines (CMH, CPH) as spin-traps and the oxygen label tetrathiatriarylmethyl to quantify O₂^•- production and concomitant oxygen consumption in human CD14⁺ monocytes. Cells were treated with PMA, fMLP and Tg which all activate NOX2 via different signalling pathways. Our results indicate that the ROS production rate after PMA stimulation is sixfold higher than that after stimulation with Tg and is independent of [Ca²⁺]_o. In contrast, Tg- induced activation depends strictly on [Ca²⁺]_o and shows a sigmoidal kinetic behaviour. Furthermore, the effect of NOX2 activators and inhibitors is related to the oxygen consumption.