Increased levels of nitric oxide (NO) at an inflammatory site may affect the biological activity of lymphoid cells. To investigate the effects of NO on the immune system, we measured the mitochondrial membrane potential (Δψm) of the peripheral blood lymphocytes (PBL) cultured with a chemical NO donor. PBL from healthy volunteers were cultured with NOC18, a NO-generating compound, at various concentrations. The Δψm of the PBL was measured by flow cytometry using 3, 3-dihexyloxacarbocyanine iodide (DiOC6(3)). NOC18 induced a decrease in the Δψm of the PBL in a dose-dependent fashion, induced an increase in the levels of reactive oxygen species (ROS), and caused these cells to undergo apoptosis. Dual-color staining of the Δψm and lymphocyte surface markers demonstrated that CD3−CD56+ natural killer (NK) cells were responsive to NO. Trolox, a vitamin E analog, partially reversed the NO-induced decrease in the Δψm of the PBL. We showed that the Δψm of peripheral NK cells were decreased by NO, which suggests that abundant NO at an inflammatory site may impair NK cell function.