Pharmacological delivery of NO stimulates the cardiac Na⁺-K⁺ pump. However, effects of NO synthesized by nitric oxide synthase (NOS) often differ from effects of NO delivered pharmacologically. In addition, NOS can become "uncoupled" and preferentially synthesize O₂^.- which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na⁺-K⁺ pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with DAF-2DA and measured fluorescence with confocal microscopy. L-Arg (500 µmol/L) increased DAF-2DA fluorescence by 51% compared with control (N=8; P<0.05). We used the whole cell patch clamp technique to measure electrogenic Na⁺-K⁺ pump current (I_p). Mean I_p of 0.35±0.03 pA/pF (N=44) was increased to 0.48±0.03 pA/pF (N=7, P<0.05) by 10 µmol/L L-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol, or NO-activated guanylyl cyclase inhibition with ODQ. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/L) induced a 51% increase in the fluorescence intensity of O₂^.- -sensitive dye DHE compared to control (N=9; P<0.05). To examine functional effects of uncoupling we measured Ip with 100 µmol/L paraquat included in patch pipette solutions. This decreased I_P to 0.28±0.03 pA/pF (N= 12; P<0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na⁺-K⁺ pump, while uncoupling of NOS causes O₂^.- mediated pump inhibition.