Oxidative DNA damage (ODD) can result from numerous endogenous metabolic processes as well as from exposure to environmental and dietary oxidants. One important type of ODD that may have a role in carcinogenesis is the formation of hydroxylated DNA bases. Our major purpose was to determine the potential for subject accrual for a multisite case-control study of ODD and breast cancer risk within a large urban university medical center. We examined the levels of a hydroxylated thymine residue, 5-hydroxymethyl-2'-deoxyuridine in DNA obtained from the peripheral blood of 26 women with breast cancer and an age-matched group of 29 control women without breast cancer. The isolated DNA was analyzed for levels of 5-hydroxymethyl-2'-deoxyuridine by gas chromatography with mass spectral detection. Our recruitment methods resulted in a relatively high yield of eligible cases (72%) and a lower yield of controls (46%). We evaluated the dose-response relationship of ODD level to breast cancer risk, using quartiles of ODD. The covariate-adjusted odds ratio of breast cancer exceeded 2.0 for women in the highest quartile of ODD (compared with the lowest quartile), although this result was not statistically significant. ODD levels were significantly more variable among African-American controls (SD = 224.1) than among white controls (SD = 57.5), p < 0.001. Overall, these results suggest a possible slight increase in breast cancer risk among women in the highest ODD quartile, after adjusting for race, menopausal status, and family history of breast cancer.