BACKGROUND Bioavailability of nitric oxide in the body may be estimated by measuring the concentration of nitrate in plasma. However, it has not been reported whether sequestering of aminothiols in plasma affects the concentration of nitrate in the samples. OBJECTIVE N-ethylmaleimide (NEM) sequesters aminothiols in plasma therefore we tested the in vivo effect of NEM on the concentration nitrate in plasma. METHODS Blood samples were collected from 56 healthy subjects in EDTA vials, EDTA vials containing PBS (pH7.4) and EDTA vials containing NEM dissolved in PBS. Nonparametric statistical tests were used to study the effect of NEM on the concentration of nitrate in plasma measured by the Griess reagent assay and by an HPLC method. RESULTS The concentration of nitrate in plasma containing NEM dissolved in PBS was lower than plasma containing PBS and plasma without any reagent measured by the Griess reagent assay (p<0.001 and p<0.001). Similarly, the concentration of nitrate in plasma containing NEM measured by the Griess reagent assay was significantly lower than nitrate concentration in plasma measured by the HPLC method (p<0.001). CONCLUSIONS Our findings suggest that plasma, which contains NEM, may not be suitable for the measurement of nitrate by the Griess reagent assay.