A novel method for the preparation of molecularly imprinted microspheres has been developed. Theophylline, caffeine, and 17 beta -estradiol are,used as template molecules to demonstrate the generally applicable methodology. In the presence of a template molecule, cross-linking polymerization starting from a dilute solution of methacrylic acid and trimethylolpropane trimethacrylate yields polymer microspheres bearing imprinted binding sites. Radioligand binding analysis confirms that very high binding specificity is retained when the molar ratio of functional monomer:cross-linker is controlled between 1:1 and 2.3:1. Nonspecific binding of molecularly imprinted microspheres decreases when an increasing amount of template molecule is used for polymer preparation. Compared with previously reported suspension polymerization and seed polymerization, the present method is highly efficient, is generally applicable to various template molecules, and gives uniform microspheres in good yield.