Background: Studies in animals and geographic correlations across populations suggest that fatty acid intake may have a positive relationship with breast cancer risk, but analytic epidemiologic studies of fat intake have been less supportive. Adipose tissue analysis provides a more objective assessment of intakes of fatty acids that are not endogenously synthesized than do the questionnaire survey methods used in many epidemiologic studies. Purpose: This case-control study of postmenopausal women was designed to examine the relationship between fatty acid composition of subcutaneous adipose tissue and risk of breast cancer and proliferative benign breast disease. In addition, we examined specific hypotheses that breast cancer risk is negatively associated with long-chain N-3 fatty acid intake, positively associated with trans fatty acid intake, and positively associated with increased intake of polyunsaturated fat together with low intake of antioxidants. Methods: Aspirates of subcutaneous fat from the buttocks were obtained from 380 women with newly diagnosed stage I or II breast cancer and 176 with proliferative benign breast disease. A total of 397 women who were evaluated for breast abnormalities at the same institutions but did not require breast biopsy or whose biopsy revealed nonproliferative benign breast disease served as the control group. We examined associations between saturated, monounsaturated, polyunsaturated, trans, or long-chain N-3 fatty acids and breast cancer, atypical hyperplasia, or proliferative benign breast disease without atypia. Results: We observed no consistent patterns of association between breast cancer risk and any of the categories of fatty acids or the individual constituent fatty acids in the adipose tissue. Saturated fatty acids were inversely associated with risk of proliferative benign breast disease without atypia but not with atypical hyperplasia or breast cancer. This association was not observed, however, when total fat intake was taken into account. Women with high levels of polyunsaturated fatty acids in adipose tissue and low serum or dietary levels of antioxidants were not observed to be at higher risk of breast cancer. Conclusions: Using an objective measure of intake, we observed no major associations between polyunsaturated fatty acids, including long-chain N-3 fatty acids and trans fatty acids, and risk of breast cancer or proliferative benign breast disease. Implications: These data do not support the hypothesis that intake of specific fatty acids, particularly polyunsaturated and trans fatty acids, is an important risk factor for malignant or benign breast disease. [J Natl Cancer Inst 85: 785–793, 1993]