Functional rat estrogen receptor beta ligand binding domain (rER beta LBD, aa 210-485) and human estrogen receptor alpha ligand binding domain (hER alpha LBD, aa 301-553) were expressed in Escherichia coli. Hormone binding assays revealed that both ER beta and ER alpha LBDs bound the natural ligand estradiol (E2) with similar affinity (Kd approximately 100 pM). Competitive binding experiments were carried out with ICI 164384, 4-hydroxytamoxifen, 16 alpha-bromo-estradiol, and genistein employing [3H]E2 as a tracer. No significant differences in responses of ER alpha and ER beta LBDs to ICI 164384 and 4-hydroxytamoxifen were observed, 16 alpha-Bromo-estradiol and genistein discriminated between the ER subtypes and acted as ER alpha and ER beta selective ligands, respectively. Final purification of recombinant proteins was achieved on an E2 affinity column, where they were subjected to in situ carboxymethylation. The partially carboxymethylated proteins actively bound E2. The carboxymethylated rER beta LBD had a molecular mass of 32251.6 Da, equivalent to the calculated mass with the addition of three carboxymethyl groups. No other proteins (of lower or higher molecular mass) were detected, so the LBD was considered structurally authentic and pure. By using a combination of intact protein mass spectrometric fragmentation and trypsin proteolysis (98% sequence coverage), it was established that rER beta cysteine-289 and -354 were not carboxymethylated on the affinity column, suggesting that they were shielded from alkylation in the E2-bound conformational state. Concurrent analysis of hER alpha LBD showed that under the same experimental conditions, the two equivalent ER alpha cysteines were not alkylated (alpha C381 and alpha C447). These data support close structural relationship between the E2-bound ER alpha LBD and ER beta LBD proteins.