Endothelium-dependent (nitric oxide [NO]-mediated) vasodilation is impaired in humans with heart failure. This dysfunction is an important therapeutic target. The plasma concentration of the NO metabolites nitrate and nitrite (collectively referred to as NOx) is a measure of whole-body NO production, provided that the dietary intake of the ions is low. Fifty clinically healthy dogs older than 1 year (median 5.0 years; interquartile interval 2.6–8.2 years) were studied, including 9 controls of various breeds, 23 Cavalier King Charles Spaniels (CKCSs) with no or minimal mitral regurgitation (MR), 9 CKCSs with mild MR (regurgitant jet occupying 15–50% of the left atrial area), and 9 CKCS with moderate to severe MR (jet ± 50%) due to myxomatous valve disease. None of the dogs received medication. The dogs were given NOx-free water and a diet with a low concentration of NOx for 96 hours before blood sampling. Multiple linear regression analysis revealed that dog group, but not gender, age, serum creatinine concentration, and platelet count, was associated with NOx concentrations. Control dogs had the same NOx concentration (median 20.0 μM; interquartile interval 15.1–25.5 μM) as CKCSs without MR (median 18.7 μM; interquartile interval 15.5–25.9 μM). Compared to CKCSs without MR, the NOx concentration was lower in CKCSs with mild (median 12.9 μM; interquartile interval 11.0–13.5 μM; P= .04) and moderate to severe (median 11.2 μM; interquartile interval 6.9–17.1 μM; P= .02) MR. In conclusion, CKCSs with mild to severe, clinically silent MR have decreased plasma NOx concentrations, suggesting that endothelial dysfunction develops early in the course of developing MR in dogs.