The mechanism by which 17β-estradiol (E2) enters uterine cells remains unclear; facilitated transport and passive diffusion have been proposed. These conflicting conclusions were drawn from experimental data obtained with intact uterine tissue, where the initial rates of estrogen uptake could not be accurately measured for a variety of reasons which will be discussed in this report. To clarify this issue, we have studied the kinetics of E2 entry into suspended uterine cells by measuring the initial rates of formation of the intracellular receptor-estradiol complex (R-E2), under conditions where the formation of cytoplasmic R-E2. and its nuclear translocation were not rate limiting. Thus, the initial rates of formation of R-E2 should be a function of the quantity of E2 which entered the cells. Data are presented demonstrating that at 37 C, E2 uptake is extremely rapid, with 20% of the intracellular receptor sites being saturated within the first 15–30 sec at extracellular E2 concentrations higher than 10 niw. When the initial rates of E2 binding at 37 and 27 C were measured within this time interval at estrogen concentrations as high as 2.3 × 10-M M, they were found to be a linear function of the estrogen concentration in the medium. Data from studies on the temperature dependence of the initial rates of estrogen binding to intact cells gave linear Arrhenius plots with an energy of activation of 16 Cal/mol. This temperature dependence may reflect the properties of the cytoplasmic estrogen receptor proteins, since the activation energy for the formation of R-E2 in uterine cytosol preparations was found to be 19 Cal/mol. Finally, the rates of E2 binding to uterine cells were not decreased by the addition of purified E2 covalently linked to bovine serum albumin. These data are interpreted to suggest that E2 enters uterine cells by passive diffusion.