Objective: To examine the effects of alcohol, caffeine, and tobacco use on early follicular phase FSH, LH, E2, and sex hormone-binding globulin (SHBG). Design: Cross-sectional study. Setting: Academic medical center. Patient(s): Four hundred ninety-eight women selected from the general population, ages 36-45, who were not currently pregnant, breast feeding, or using exogenous hormones. Intervention(s): A general questionnaire assessing demography, anthropometry, and smoking habits and a standardized dietary questionnaire assessing food and beverage frequencies, including sources of alcohol and caffeine. Main Outcome Measures: FSH, LH, E2, and SHBG levels measured during the early follicular phase of the menstrual cycle. Result(s): Significant associations observed in a univariate analysis included age ≥40 and current smoking associated with higher FSH; higher body mass index (BMI) associated with lower SHBG levels; and daily alcohol use, cholesterol consumption greater than the median, and coffee use >1 cup/d associated with higher E2 levels. In a multivariate model, total caffeine use was significantly associated with E2 levels after adjustment for age, BMI, total calories, current smoking, alcohol, cholesterol consumption, and day of sampling. Early follicular phase E2 increased from 28.2 pg/mL for women consuming ≤100 mg of caffeine to 45.2 pg/mL for women consuming ≥500 mg of caffeine per day, about a 70% increase. Conclusion(s): Coffee consumption and total caffeine use may increase early follicular phase E2 levels independent of related habits of alcohol or tobacco use.