The association between serum lipids and breast cancer risk was investigated in a cohort of 5,207 Danish women, who participated in The Glostrup Population Studies between 1964 and 1986. During four to 26 years of follow-up, 51 incident cases of breast cancer were identified by linkage to the Danish Cancer Registry. At the time of lipid measurement, the women were between 30 and 80 years of age. An inverse association was found between serum high-density lipoprotein (HDL) cholesterol and risk of breast cancer, which was not changed by adjustment for potential confounders such as social class, age at menarche and menopause, number of full-term pregnancies, body mass index, or alcohol and coffee consumption. The relative risk was 0.3 (95 percent confidence interval = 0.1-0.8) for women in the highest quartile of serum HDL-cholesterol compared with women in the lowest quartile and the relation displayed a significant negative trend (P = 0.01). For serum triglycerides there was a suggestion of a positive association with breast cancer incidence, but the trend was not significant (P = 0.06). No relationship between total serum cholesterol or low-density lipoprotein cholesterol and risk of breast cancer was observed. Risk estimates for well known breast cancer risk factors such as social class, age at menopause, number of full-term pregnancies, and obesity were in the directions expected.