Aim. Nitric oxide (NO) is an important molecule with major functions in the regulation of the vascular tone. NO is also important for the deformability of human red blood cells (hRBCs), which secures a sufficient supply of the microenvironment with oxygen (O2). Therefore, the aim of this study was to assess the production of NO in hRBCs as there is a great interest in the NO pool during different diseases and the effects of certain treatments (e.g. exercise) on this pool. Different approaches determine NO in cells: (1) oxidation products of NO (e.g. nitrite or nitrate) and (2) diaminofluorescein-diacetate (DAF-2DA). DAF-2DA is a cell-permeable agent and reacts in the presence of O2 to the highly fluorescent derivate triazolofluorescein (DAF-2T). Methods. Blood samples were taken from healthy males before and directly after a 60 min lasting extensive (70% of VO2max) run. 100mL of blood were immediately equilibrated with 2mL DAF-2DA (c = 10mM) for 3 min. After equilibration, 50mL of blood were used for analyses. NO inhibitors (e.g. L-NAME and Wortmannin) were used to block the signal to assure that an NO signal was detected. The chemically loaded hRBCs were imaged on a confocal laser scan microscope. Results. Under basal conditions there was no NO signal detectable in hRBCs after equilibration with DAF-2DA. In contrast, exercise increased the pool of NO in hRBCs as detected as described. By administration of NO inhibitors the NO specific fluorescent signal could be diminished. Conclusion. The increase in NO after exercise might be related to elevated shear stress in hRBCs, which is a potent inducer of NO. The results show that DAF-2DA is a useful tool to assess the NO pool in hRBCs beside the assessment in ECs as described earlier. It might provide further insights into the contribution of hRBCs to the peripheral vascular tone and to the deformability capacities of hRBCs in different diseases. Further research is needed to make the fluorescent signal quantifiable.