An efficient novel method for the synthesis of a covalent molecularly imprinted polymer (MIP) highly specific to beta-estradiol have been developed. MIP prepared by both covalent and non covalent techniques, demonstrated high selectivity toward beta-estradiol. MIPs were synthesized by radical polymerization of 17-beta-estradiol 4-vinyl-benzene carboxyl or sulfonyl esters used as covalent functional monomers, methacrylic acid as noncovalent functional monomer, ethylene glycol dimethacrylate as crosslinking agent, and acetonitrile as swelling and porogenic component. Almost 35% (w/w) of 17-beta-estradiol was successfully removed from the polymer network by basic hydrolysis. The binding ability of MIP was 10.73 mu g/mg MIP following removal of 17-beta-estradiol in the 2 mg/mL beta-estradiol solution. Selective rebinding of beta-estradiol toward MIP was tested in the presence of competitive binders including estrone, 19-nortestosterone, epiandrosterone, and cholesterol. Estrone having closest similar chemical structure to beta-estradiol exhibited only 0.6 mu g/mg MIP competitive binding, being exposed to equivalent concentrations. Moreover, other competitive steroids demonstrated negligible affinity toward MIP indicating high selectivity of novel MIP system toward beta-estradiol. (C) 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5534-5542, 2009