Poly(HEMA) microbeads were prepared by suspension polymerization of 2-hydorxyethylmethacrylate and ethyleneglycoldimethacrylate (EGDMA). The water content, ligand density, and selectivity for poly(His)-tagged D-hydantoinase of the poly(HEMA)-based adsorbents were affected by the concentration of EDGMA used during polymerization. The Ni(II)-loaded poly(HEMA) adsorbent exhibited an adsorption capacity of 1.0 mg/g for poly(His)-tagged D-hydantoinase under optimal conditions with buffer containing 100-300 mM NaCl at pH 6.0. One-step purification protocol with the adsorbent gave a purity of at least 92%. The adsorption capacity of adsorbent declined by 54% after 7 cycles, due to the leaching of Ni(11) from the adsorbent. However, upon regeneration the adsorption capacity can be restored. Given the ease of preparation and the chemical and microbial resistance, the poly (HEMA)-based IMAC adsorbent could be a promising substitute for the polysaccharide-based IMAC adsorbents. (c) 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.