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The steroid 17beta-estradiol (E2) acts to modulate transcription through classical nuclear estrogen receptors (ER-alpha and ER-beta). However, E2 also induces a number of rapid responses (<10 min) within cells, including cells devoid of classical ERs, consistent with the presence of a membrane receptor for E2. Membrane impermeable steroids, typically bovine serum albumin (BSA) conjugates, are commonly used to characterize these non-genomic actions of E2 to exclude the involvement of nuclear ERs. Here we report that E2-BSA conjugate preparations, but not unconjugated E2, activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the SK-N-SH neuroblastoma cell line, raising concerns regarding the use of these reagents as E2 mimics. Freshly prepared solutions of E2-BSA were found to contain free immunoassayable E2 (iE2), which could be removed by filtration. E2-BSA solutions devoid of free iE2 failed to compete for binding of 125I16alpha-iodo-E2 to ER-alpha or ER-beta. Furthermore, in contrast to E2, E2-BSA conjugates did not bind to ER-alpha or ER-beta as assessed by electrophoretic mobility shift analyses. Protein analysis demonstrated that certain E2-BSA preparations were of very high molecular weight, suggesting extreme protein cross-linking. These findings suggest that E2-BSA does not mimic E2 and is not an appropriate ligand for investigating estrogen receptors. This underscores the need to design stable, cell impermeable analogs of estrogen for the characterization of membrane estrogen receptors.