The question of specific immunomodulating qualities of hypertonic saline (HTS) has not been settled. It has proven difficult to distinguish between immunomodulation directly attributable to HTS and influence because of favorable circulatory effects. The nature of immune activator may also play a role. In a whole-blood model, we have investigated these relations further, with special emphasize on osmolalities usually found after recommended dosing. Blood from 10 healthy donors was exposed to osmolalities ranging from 295 to 480 mOsm/kg and stimulated with the two clinically relevant stimulators peptidoglycan (1 µg/mL) or LPS (10 ng/mL) for 6 h at 37°C. Leukocyte response was evaluated by measuring selected cytokines in the supernatant. Moderate hyperosmolality alone boosted the release of CXCL8/IL-8. The peptidoglycan-stimulated synthesis of pivotal proinflammatory cytokines was inhibited in an osmolality-dependent way, but statistically significant only at osmolalities above those attained after routine use of HTS, i.e., 310 mOsm/kg or greater: IL-6 (P < 0.05 at 315 mOsm/kg), IL-1ß, and TNF-α (P < 0.05 at 335 mOsm/kg). Similar effects were seen for the chemokine CCL3 and the anti-inflammatory cytokine IL-10. In contrast, the effects in cells stimulated with LPS were either lower or absent. Thus, osmolalities usually found after clinical use of HTS only modestly influenced the selected immune parameters, regardless of stimulator.