Background: Elevated pregnancy hormone levels, such as oestrogen and progesterone, may increase the risk of developing breast cancer both in mothers and offspring. However, the reasons for large inter-individual variations in estrogen and progesterone levels during pregnancy remain unknown. The objectives of this study were to investigate whether a) intakes of total dietary fat, types of fat (monounsaturated: MUFA, polyunsaturated: n-3 and n-6 PUFA, and saturated) and b) gestational weight gain are associated with estradiol and progesterone levels in plasma during pregnancy. Methods: We measured body weight as well as estradiol and progesterone in plasma among 226 healthy pregnant Swedish women on gestation weeks 12, 25 and 33. At the same time points, dietary intake of total fat and types of fat (MUFA, PUFA, SFA, n-3 and n-6 PUFA) were estimated using 3-day food diaries. Results: A large variation in estradiol and progesterone levels was evident. Nulliparous women had 37%, 12% and 30% higher mean estradiol levels on gestation weeks 12, 25 and 33 compared to parous women (P = 0.008). No associations were found between dietary intake of total fat or fat subtypes (including n-3 PUFA and n-6 PUFA) and plasma estradiol or progesterone levels. Gestational weight gain was associated with progesterone levels (P = 0.03) but the effect was very small (20% increase in progesterone levels between gestational weeks 12 and 33 per kg body weight/week). Conclusion: No associations among gestational weight gain, maternal dietary fat intake (total or subtypes including n-3 PUFA and n-6 PUFA) and plasma estradiol levels were found. However, pregnancy progesterone levels correlated with weight gain during pregnancy. Identification of other possible determinants of pregnancy estradiol and progesterone levels, important for the development of breast cancer in both mothers and offspring, are needed.