The initial event in the life cycle of a virus is its interaction with specific receptors present on the surface of a cell. Understanding these interactions is important to our understanding of viral tropism and tissue specific pathology associated with viral disease. The human polyomavirus, JCV, is the etiological agent of the fatal central nervous system (CNS) demyelinating disease, progressive multifocal leukoencephalopathy (PML). PML is the direct result of JCV infection of oligodendrocytes, the myelin producing cell in the CNS. In vivo, JCV can be detected in oligodendrocytes, astrocytes, lymphoid tissue, and peripheral blood of PML patients. In vitro, JCV infects human glial cells, tonsilar stromal cells, and, to a limited extent, human B lymphocytes. The initial step in infection of cells by JCV is at the level of attachment and entry. A specific cell surface receptor for JCV on human glial cells has not been identified. To begin to understand the nature of JCV receptors on human glial cells, large quantities of a previously characterized hybrid JC virus (Mad-1/SVEdelta) were purified. A direct virus binding assay demonstrated that these highly purified and labeled JCV virions bound to a finite number of cellular receptors on human glial cells. A competitive virus binding assay demonstrated that an excess of unlabeled JCV competed with labeled JCV more efficiently than did an excess of purified SV40. Furthermore, anti-class I antibodies which inhibited infection of glial cells by SV40 had no significant effect on infection by JCV. These results imply that JCV does not share receptor specificity with the related polyomavirus, SV40.