In the presence of a template molecule, transaconitic acid and, using acetonitrile as solvent and dispersing medium, monodispersed microspheres with a diameter of 600-700 nm bearing molecularly imprinted binding sites were prepared by precipitation polymerization. It was found that the concentrations of template, monomer, and crosslinking agent as well as the chemical structure of the template greatly affect the polymer configuration. Microspheres are produced only when the concentration of the template molecule and the functional monomer are finely tuned. Comparison with the performance of a conventional imprinted polymer monolith showed that the imprinted microsphere had obvious advantages in specific binding to template molecule. (C) 2004 Wiley Periodicals, Inc.