Early stages of infection by the mouse polyomavirus have been studied using HeLa cells stably expressing small interfering RNA to protein disulfide isomerase (PDI). Infectibility measured by nuclear T antigen expression was reduced commensurately with the degree of PDI downregulation. Infectibility was restored by transfection with a plasmid expressing PDI but not with a control expressing catalytically inactive enzyme. Deconvolution microscopy using fluorescently labeled virus and cellular markers showed that virus reaches the endoplasmic reticulum (ER) normally in cells with reduced PDI but subsequently fails to exit the ER. Simian virus 40 infection was not inhibited in PDI-downregulated cells. The results are discussed in terms of structural differences between the two viruses and current knowledge of virus disassembly in the ER.