Nitrotyrosine (NO2Y) is a global marker of protein modification by reactive nitrogen species such as peroxynitrite derived from nitric oxide (NO). Because NO and its derivatives are postulated to enhance carcinogenesis, we used stable isotope dilution mass spectrometry to measure the levels of NO2Y in 30 samples of human breast cancer of varying pathologic types. In the samples tested, the NO2Y levels were generally low (average of 14.1 +/- 9.2 micromol NO2Y per mole of tyrosine). Breast cancers with a high microvascular density, however, had a significantly higher average level of NO2Y than tumors with a low microvascular density (20 v.s. 10 micromol NO2Y per mole of tyrosine, p = 0.007 by two-tailed t-test, assuming unequal variances of two samples). There was no apparent association between NO2Y levels and the differentiation of the tumors, tumor aneuploidy, estrogen receptor status, HER-2 expression, lymph node status, or infiltration of the tumors by neutrophils or eosinophils. When the tissues were stained by immunohistochemistry for NO2Y, the NO2Y was localized predominantly within inflammatory cells located immediately adjacent to blood vessels at the edges of the tumors. NO2Y was generally not evident within the tumor cells or inflammatory cells in the stroma. We conclude that low levels of reactive nitrogen species are located predominantly within inflammatory cells near blood vessels of breast cancer and that higher NO2Y levels are associated with an increased density of blood vessels. Our findings, therefore, support a possible association between inflammatory cells and reactive nitrogen species in modulating microvascular density at the edges of breast cancer.