Diminished nitric oxide (NO) bioactivity and enhanced peroxynitrite formation have been implicated as major contributors to atherosclerotic vascular dysfunctions. Hallmark reactions of peroxynitrite include the accumulation of 3-nitrotyrosine (3-NT) in proteins and oxidation of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH4). The present study sought to: (i) quantify the extent to which 3-NT accumulates and BH4 becomes oxidized in organs of ApoE^-/- atherosclerotic mice, and (ii) determine the specific contribution of inducible NOS (iNOS) to these processes. Whereas protein 3-NT and oxidized BH4 were undetected or near the detection limit in heart, lung and kidney of 3-week old ApoE^-/- mice or ApoE^-/- mice fed a regular chow diet for 24 weeks, robust accumulation was evident after 24 weeks on a Western (atherogenic) diet. Since 3-NT accumulation was diminished 3- to 20-fold in heart, lung and liver in ApoE^-/- mice missing iNOS, iNOS-derived species are involved in this reaction. In contrast, iNOS-derived species did not contribute to elevated protein 3-NT formation in kidney or brain. iNOS deletion also afforded marked protection against BH4 oxidation in heart, lung and kidney of atherogenic ApoE^-/- mice, but not brain or liver. These findings demonstrate that iNOS-derived species are increased during atherogenesis in ApoE^-/- mice and that these species differentially contribute to protein 3-NT accumulation and BH4 oxidation in a tissue-dependent manner. Since BH4 oxidation can switch the predominant NOS product from ·NO to superoxide, we predict that progressive NOS uncoupling is likely to drive atherogenic vascular dysfunctions.