Paraoxonase-1 (PON-1) is a high-density-lipoprotein-bound enzyme, and its major function is to prevent oxidation of low-density lipoprotein. Atherogenesis could be related to decreased activity of this enzyme. Nitrites (NO2-), either present as a contaminant and/or the main metabolic end product of nitric oxide (NO) degradation, may trigger nitrative damage to PON-1 enzyme. Minimal information is available concerning the effect of nitrite on the enzyme activity and the mechanism which it exerts its effect. The aim of this study was to analyze whether nitrites could play a role in modifying human PON-1 activity. Our results revealed that PON-1 activity was inhibited by nitrite in dose- and time-dependent manner. Site-specific nitration focused on phenolic residues, particularly tyrosine residues of the enzyme, may result in modification of its biological functions. Nitration of phenolic residues occurs via peroxynitrite (ONOO-) formation, which requires peroxides and nitrite. Thus, we tested the presence of peroxides, which are found in all plasma samples regardless of nitrite concentration. The inhibition of PON-1 activity by nitrite was significantly reduced by tryptophan, reduced glutathione (GSH), and catalase additions. Therefore, we concluded that nitrites may have a role in the inactivation of PON-1, probably through nitration of enzyme phenyl residues, and additions of individual aromatic amino acids, with highlighting on tryptophan, could be of important value in minimizing the nitrite-induced inhibition of PON-1 enzyme.