Challenge studies of microfiltration (MF) and ultrafiltration (UF) membranes were conducted using two fluorescent microspheres (26 and 67 nm) and two bacteriophage (MS2 and PRDI), 0.22 mu m hydrophilic and hydrophobic polyvinylidene fluoride (PVDF) MF membranes, a 100 kD hydrophilic regenerated cellulose (RC) UF membrane, and a 100 kD hydrophobic polyethersulfone (PES) UF membrane. The membranes, bacteriophage, and microspheres were fully characterized according to their surface properties, including hydrophobicity, isoelectric point, surface charge, diameter of the phage or sphere, and membrane pore size. Microspheres were found to be poor surrogates for bacteriophages at the bench scale in testing UF membranes, but adequate in testing MF membranes with lower microbial rejection. The findings presented here have implications for use of microspheres as surrogates for other microorganisms. If surface charge and other characteristics are not properly considered, using microspheres as surrogates for microorganisms may yield inconsistent results where the rejection mechanism is by adsorption, adhesion, or where electrostatic considerations are important, rather than by simple size exclusion. (C) 2009 Elsevier B.V. All rights reserved.