A physiologically based pharmacokinetic (PBPK) model for estradiol (E2) in rats and humans (male and female) was developed to provide a quantitative tool for evaluating the importance of physiological parameters on E2 blood and tissue concentration time-course and for predicting blood and tissue concentrations in rats and humans. A hepatic extraction model was developed to evaluate the significance of plasma protein binding on the hepatic extraction of E2 and the approach was integrated into the E2 model. Sufficient data was available to parameterize and validate oral and iv routes. The E2 model simulations of E2 blood and tissue concentrations compared well to experimental values. Estrogen receptor content strongly impacts distribution and elimination kinetics of E2 as well as tissue concentrations. The prolonged terminal elimination phase seen after iv bolus administration reflects the slow release of receptor bound E2 from tissues. E2 uptake behavior in the ovariectomized, but not intact rat uterus, was best described as diffusion-limited. Simulations with the hepatic extraction model predicted extensive binding of E2 to albumin (rat) and SHBG (sex-hormone binding globulin humans), although hepatic extraction does not appear to be restricted to the unbound fraction, implying that the total plasma E2 concentration is important when considering hepatic uptake. Important determinants of E2 disposition are tissue ER content and binding affinity, nonreceptor binding proteins, vascular permeability, partition coefficients, hepatic blood flow, and extrahepatic metabolism. As an integral part of a research program, the quantitative framework developed for E2 can be extended to other endocrine-active compounds (EACs) and used to evaluate the biological activity of EACs.