The capacity of virus filters used in the purification of therapeutic proteins is determined by the rate and extent of membrane fouling. Current virus filtration membranes have a complex multilayer structure that can be used with either the skin-side up or with the skin-side facing away from the feed, but there is currently no quantitative understanding of the effects of membrane orientation or operating conditions on the filtration performance. Experiments were performed using Millipore's Viresolve 180 membrane under both constant pressure and constant flux operation with sulfhydryl-modified BSA used as a model protein. The capacity with the skin-side up was greater during operation with constant flux and at low transmembrane pressures, with the flux decline or pressure rise due primarily to osmotic pressure effects. In contrast, data obtained with the skin-side down showed a slower, steady increase in total resistance with the cumulative filtrate volume, with minimal contribution from osmotic pressure. The capacity with the skin-side down was significantly greater than that with the skin-side up, reflecting the different fouling mechanisms in the different membrane orientations. These results provide important insights for the design and operation of virus filtration membranes.