Abstract Introduction The purpose of the study was to elucidate the association between smoking and estradiol (E2) and testosterone (T) levels in young healthy women from high-risk families. Cigarette smoke contains multiple carcinogens and is considered a risk factor for breast cancer. However, tobacco also contains aromatase inhibiting substances, but the impact on hormonal levels in young women at the age when breast cancer is initiated is unclear and needs further elucidation. Material and methods Between 1996 and 2002, 258 healthy women from high-risk breast cancer families in Sweden were enrolled in a study on the impact of lifestyle factors in women <=40 years. Blood samples and body measurements were obtained in the luteal phase (5-10 days before predicted onset of the next menstrual cycle) by a trained research nurse. Participants filled out questionnaires on lifestyle and reproductive factors. Ongoing breastfeeding, use of hormonal contraceptives other than combined E+P oral contraceptives (OCs), as well as missing data on onset of next menstrual cycle and smoking status led to exclusion of 28 women. Two hundred thirty women including 33 known BRCA1/2 mutation carriers were included in the analyses. Weight and hormonal levels were not normally distributed and were transformed using the natural logarithm to obtain better distribution. Since OCs influence endogenous hormones, the women were stratified by current OC use. Multivariable linear regression were used to obtain P-values, adjusted for age, nulliparity, menstrual cycle day, waist-to-hip ratio (WHR), and weight. Geometric means for current smokers and non-smokers were standardized at age 29 years, nulliparity, number of days until onset of next menstrual period (7 days), WHR 0.76, and LNweight (64 kgs). Results In all women, the median age was 29 years old, (IQR 24-35), and the median year of birth was 1969, the median weight was 64 kgs, and the median WHR was 0.76. In total, 47% were parous, and 42% had ever smoked, whereas 22% were current smokers, and 38% were current OC users (40% of the smokers and 37% of the non-smokers). In non OC users, E2 levels were similar in smokers and non-smokers (adjP=0.26), while standardized total T levels were higher in smokers (51.8 vs 45.1 ng/dL; adjP =0.067). In contrast, among current OC users, smokers had higher standardized E2 levels (22.7 vs 17.7 pg/mL; adjP =0.023) compared with non-smokers, while total T levels were similar in smokers and non-smokers (adjP=0.56). Conclusion In non-OC users, a more androgenic profile was observed in smokers compared with non-smokers. While in OC users endogenous E2 levels were higher in smokers compared with non-smokers, which suggests that cigarette smoke may reduce the efficacy of the pill. Differential impact of smoking on endogenous E2 and total T levels in current OC users and non-users was thus observed. Therefore, history of OC use needs to be incorporated in studies on associations between smoking and breast cancer risk. Citation Format: Carolina Ellberg, Håkan Olsson, Helena Jernstrom. Smoking impacts endogenous estradiol and testosterone levels in young healthy women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4263. doi:10.1158/1538-7445.AM2017-4263