We will describe the identity and function of two unexpected estrogen binding proteins from rat brain cell membranes in search for the putative membrane estrogen receptor (mER). An E-6-BSA column retained a distinctive 37-kDa protein that showed 100% homology with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A P-3-BSA column also retained the same protein but with less affinity. E-6-BSA bound to GAPDH with an IC50 of 50 nM, whereas the IC50 for P-3-BSA was about 500 nM. A dose of 10 nM 17beta-estradiol stimulated the catalysis of GAPDH, whereas progesterone at 100 nM inhibited it. Other steroids were ineffective. We examined if GAPDH activity would change during the rat estrous cycle, and what would be the effect of ovariectomy and estrogen treatment. The hippocampus and cerebellum were collected and GAPDH catalysis in both cytosolic and plasmalemmal-microsomal fractions was tested. The highest activity was found in Proestrus morning and the lowest in Estrus in both fractions. After ovariectomy (3 weeks) the hippocampus membrane fraction showed significantly reduced activity compared to that of Diestrus. An injection of estradiol in ovariectomized rats (10 microg/rat, s.c.) increased GAPDH activity in the hippocampus membrane fractions close to 60% from that of ovariectomized oil-treated controls 24 h after treatment maintaining similar levels by 48 h. No changes were detected in the preparations from the cerebellum of the same rats. The other protein retained by E-BSA columns was a 55-kDa protein identified as beta-tubulin. Two other proteins were also co-purified from the rat hippocampus: a 37-kDa (GAPDH) and a 45-kDa (actin). A purified brain tubulin (Cytoskeleton) was also retained with high affinity by the E-6-BSA, but with less affinity by an E-17-BSA column and not retained by either BSA, P-3-BSA or C-21-BSA columns. E-6-[125I]BSA bound with high affinity to tubulin (1 microg) and 17beta-estradiol completely displaced the binding at 10(-7) M. 17alpha-estradiol was ineffective and neither progesterone, corticosterone, DES nor 2-methoxyestradiol (2-ME) was able to displace the ligand. The T-3-[125I]BSA also bound to tubulin. But it seems to interact with another binding site, because colchicine at 10(-5) M completely eliminated the binding of T-3-[125 I]BSA to tubulin but did not displace the E-6-BSA site. Taxol competed off both ligands but only by 50%. None of the two ligands bound actin. These novel findings add new information to be considered in the intracellular actions of estradiol, particularly in the remodeling and functions of the cytoskeleton.