In this study, acetylcholinesterase (AChE) and choline oxidase (ChO) were co-immobilized on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes with the aim of using them in biosensor construction. pHEMA membranes were prepared with the addition of different salts in different HEMA : aqueous solution ratios and characterized in terms of porosity, thickness, permeability, and mechanical properties. Membranes prepared in the presence of SnCl4 were found to be superior in terms of porosity and permeability and were chosen as the immobilization matrix. Immobilization of the enzymes was achieved both by entrapment and surface attachment via epichlorohydrin (Epi) and Cibacron Blue F36A (CB) activation. The effect of immobilization on enzyme activity was evaluated by the comparison of K-m and V-max values for the free and immobilized bi-enzyme systems. The increase in K-m was negligible (1.08-fold) for the bi-enzyme system upon immobilization on surface but was 2.12-fold upon entrapment. Specific activity of the free enzyme system was found to be 0,306 mV s(-1) mug(-1) ChO while it was 0.069 (4.43-fold decrease) for entrapped and 0.198 (1.54 fold decrease) for CB-Epi immobilized enzymes. The performance of immobilized enzymes in different buffer types, pH, and temperature conditions were evaluated. The best enzyme activity was obtained at pH 9.0. Activity of the enzymes was found to increase with increasing temperature (in the range 25-40degreesC).