Virus filtration can provide a robust method for removal of adventitious parvoviruses in the production of biotherapeutics. Although virus filtration is typically thought to function by a purely size-based removal mechanism, there is limited data in the literature indicating that virus retention is a function of solution conditions. The objective of this work was to examine the effect of solution pH and ionic strength on virus retention by the Viresolve(®) NFP membrane. Data were obtained using the bacteriophage ϕX174 as a model virus, with retention data complemented by the use of confocal microscopy to directly visualize capture of fluorescently labeled ϕX174 within the filter. Virus retention was greatest at low pH and low ionic strength, conditions under which there was an attractive electrostatic interaction between the negatively charged membrane and the positively charged phage. In addition, the transient increase in virus transmission seen in response to a pressure disruption at pH 7.8 and 10 was completely absent at pH 4.9, suggesting that the trapped virus are unable to overcome the electrostatic attraction and diffuse out of the pores when the pressure is released. Further confirmation of this physical picture was provided by confocal microscopy. Images obtained at pH 10 showed the migration of previously captured phage; this phenomenon was absent at pH 4.9. These results provide important new insights into the factors governing virus retention using virus filtration membranes.