A fluorescent estradiol macromolecular complex was used to study and to characterize steroid binding to membranes of living target cells. Ligand binding to plasma membranes was quantitated with a sensitivity of 0.1 nM. In this way, we found two types of estradiol-binding sites on hormone sensitive MCF-7 cells. Type A sites (8000-16000 sites per cell) were rapidly saturated at low concentrations of the estradiol-bovine serum albumin-fluorescein isothiocyanate macromolecular complex (E2-BSA-FITC). They had a greater affinity for the complex than did the type B sites for which a phenomenon of cooperative fixation was shown. The complex binding was displaced by estrogenic molecules, but not by non-estrogenic compounds, such as cortisol or progesterone. We also studied complex binding on another breast cancer cell line, MDA-MB-231 (MDA), without intracellular estrogen receptors. These cells showed a specific plasma membrane binding system for estrogen, but lacked the high affinity type A binding site. Then, we report the effects of enzyme treatments (trypsin, phospholipase A2 and neuraminidase) on E2-BSA-FITC binding to MCF-7 cell membranes. The quantity of complex bound to membranes decreased after phospholipase and neuraminidase treatments and increased after trypsin. But, in the three cases, the binding was no longer specific because it could not be displaced by E2-BSA or by estradiol. The enzymatic effects were reversible and specific binding was totally restored within 24 h. However, in the presence of the protein synthesis inhibitor, cycloheximide, no restoration of specific binding occurred on trypsin-treated cells. Estrogen binding to MCF-7 and MDA cell plasma membranes thus possesses the three characteristics of all mediated transport processes across biological membranes: saturability, substrate specificity, and specific inhibition. However, the high affinity type A binding site was found only on the estrogen-sensitive cell line, MCF-7.