The objective of this study was to identify the optimal timing of sampling during the menstrual cycle for assessment of interindividual variation in exposure to endogenous sex hormones, including estradiol, progesterone, and the free androgen index. Twenty-four healthy premenopausal women with regular periods were recruited, and alternate day venous blood samples were taken in the morning throughout one menstrual cycle. Spearman rank correlation coefficients were calculated for the estimates of average hormone levels (based on area under the curve) over one menstrual cycle against values on single days within that cycle. Days within the menstrual cycle were identified that provided the best assessment of interindividual differences. The most consistent correlation for estradiol was seen between days 9 and 11 (e.g., r = 0.53 and P = 0.01, day 10), the most consistent correlation for progesterone was seen between days 17 and 21 (e.g., r = 0.80 and P < 0.001, day 20), and the most consistent correlation for free androgen index was seen between days 12 and 15 (e.g., r = 0.90 and P < 0.001, day 15). Post hoc analysis of estradiol and progesterone levels on days counted back from the start of the next menstrual cycle identified marginally stronger associations. On repeat hormone measurements (not done for progesterone) on days 10 and 15, two to five menstrual cycles later, correlation coefficients with the original hormone levels remained reasonable (> or =0.55) for most. In conclusion, a reasonable characterization of interindividual differences in premenopausal estradiol, androgen, and progesterone levels may be achieved with single blood samples taken on specific days. This provides a useful approach for future epidemiological studies.