Semicrystalline poly(vinyl alcohol) (PVA) ultrafiltration (UF) membranes were fabricated for use in bioseparations to separate small molecular weight solutes from macromolecules. PVA was chosen because of its hydrophilic nature and these membranes are expected to minimize membrane fouling due to protein adsorption. The membranes were annealed at temperatures above the polymer's glass transition temperature to crystallize them in order to improve their mechanical properties. The degree of crystallinity of these membranes was measured using differential scanning calorimetry. Studies were conducted to measure the selectivity of separation of two different sized solutes using these membranes. These studies showed that heat-treated PVA membranes had a higher selectivity compared to the untreated PVA membranes. The effects of heat treatment, stirring speed, membrane thickness, average molecular weight of PVA and the addition of Pluronic(R) F127 and Pluronic(R) F68 on the separation of fluorescein isothiocyanate-dextran and myoglobin from L-tryptophan were studied. The mechanical strength of heat-treated PVA membranes and the selectivity of separation of these membranes were compared with commercial polyethersulfone (PeS) membranes. Mechanical studies using dynamic mechanical analysis showed that the tensile modulus of commercial PeS membranes was significantly higher than the heat-treated PVA membranes that were swollen in deionized water for 1 and 5 h. However, the compressive moduli of PVA membranes were found to be of the same order as the compressive moduli of PeS membranes. The tensile and compressive moduli of heat-treated PVA/1% (w/v) Pluronic(R) F127 membranes were considerably lower than PeS and PVA membranes. It was found that the PVA membranes were more resistant to fouling than the PeS membranes.