To examine the function of JC virus (JCV) agnoprotein, we examined the brains of cases of progressive multifocal leukoencephalopathy (PML), which is caused by JCV infection, using a newly generated antibody. The antibody reacted with 8 kDa protein specific for JCV agnoprotein by Western blotting. In vitro analyses showed that JCV capsid protein VP1 and large T antigen (T-Ag) were localized in the nuclei, but that agnoprotein was mainly detected in the cytoplasm of JCV-infected cells with an occasional nuclear staining. In the PML brain, an immunoreactive signal for agnoprotein was distributed in the perinuclear areas and cytoplasmic processes with occasional punctate staining in demyelinating lesions as well as adjacent myelinated areas. Agnoprotein presented mostly in the infected oligodendrocytes and partly in the astrocytes. Using double immunostaining, agnoprotein was seen to be expressed in the cytoplasmic processes of the cells, the nuclei of which were labeled with VP1 and T-Ag, where virus particles existed. Thus, JCV agnoprotein was mostly expressed in the infected oligodendrocytes and mainly localized in the cytoplasmic processes apart from virus particles in the demyelinated lesions.