The N-methyl d-aspartate receptors (NMDARs) mediating Ca(2+) uptake upon stimulation with glutamate and glycine were recently discovered in red blood cells (RBC) of healthy humans. Activation of these receptors with agonists triggered transient Ca(2+)-dependent decrease in hemoglobin oxygen affinity in RBC suspension. The aim of this study was to assess the potential physiological relevance of this phenomenon. Two groups formed by either healthy untrained volunteers or endurance athletes were subjected to a stepwise incremental cycling test to exhaustion. Plasma glutamate levels, activity of the NMDARs, and hemoglobin O2 affinity were measured in blood samples obtained before and after the exercise in both groups. Increase in plasma glutamate levels following exercise was observed in both groups. Transient Ca(2+) accumulation in response to the NMDAR stimulation with NMDA and glycine was followed by facilitated Ca(2+) extrusion from the RBC and compensatory decrease in cytosolic Ca(2+) levels. Short-term activation of the receptors triggered a transient decrease in O2 affinity of hemoglobin in both groups. These exercise-induced responses were more pronounced in athletes compared to the untrained subjects. Athletes were initially presented with lower basal intracellular Ca(2+) levels and hemoglobin oxygen affinity compared to non-trained controls. High basal plasma glutamate levels were associated with induction of hemolysis and formation of echinocytes upon stimulation with the receptor agonists. These findings suggest that glutamate release occurring during exhaustive exercise bouts may acutely facilitate O2 liberation from hemoglobin and improve oxygen delivery to the exercising muscle.