Methods are described for the measurement of the estradiol-binding capacity of TeBG and of the free, TeBG-bound, and non-specifically protein-bound fractions of plasma estradiol. Each determination used undiluted plasma at 37 C, and a total volume of less than 2.0 ml of plasma was required to complete all the assays. The measurement of the per cent of free estradiol was affected by changes in plasma dilution. The measurement of the other fractions of estradiol was not influenced by changes in either the dilution or the volume of plasma. The distribution of plasma estradiol was determined daily throughout 5 individual menstrual cycles. The per cent of free, the per cent of TeBG-bound, and the TeBG binding capacity of estradiol remained constant throughout the cycle with mean values of 2.21 +/- 0.04% (SE), 38.4 +/- 0.7%, and 16.6 +/- 0.43 ng/ml, respectively. The mean association constant of TeBG for estradiol was 6.58 +/- 0.25 x 10(7)M-1. The concentration of the free and non-specifically protein-bound fractions of estradiol paralleled the total plasma concentration of estradiol. The results show that biologic events related to normal cyclic changes of plasma estradiol may be attributed to fluctuations in the free estradiol and to estradiol which is bound with low affinity to non-specific plasma proteins.