Unique receptor sites for poliovirus are considered to be the primary determinant of the virus' cell and tissue-type specificity. To study the poliovirus-cell interaction, eight monoclonal antibodies that specifically block the cytopathic effects of poliovirus were generated by using HeLa cell preparations as immunogen and a newly developed colorimetric screening assay. Plaque-inhibition assays confirmed the viral specificity of the antibodies, and when one antibody, AF3, was used as a probe in immunoblots of cell membrane preparations, it detected a 100-kDa band in only those cell lines and tissues permissive for poliovirus infection. AF3 also specifically inhibited radiolabeled poliovirus binding to cells. In terms of tissue specificity, AF3 detected the 100-kDa band in membrane preparations from human spinal cord but not in organ homogenates of human kidney or in murine tissue, including the central nervous system. Furthermore, AF3 detected the band in a human-mouse hybrid cell line containing human chromosome 19, which confers permissivity for poliovirus infection, but the antibody did not detect the band in a human chromosome 19-deficient subclone. In an immunohistochemical study of the human brainstem, AF3 stained neurons in the reticular formation and clusters of brainstem neurons, consistent with the known pattern of damage caused by poliovirus infection in the brainstem. Furthermore, AF3 reacted with human peripheral mononuclear cells, consistent with the known replication of poliovirus in Peyer's patches and tonsils. These results strongly suggest that the 100-kDa band detected by antibody AF3 is, or is closely associated with, the poliovirus receptor site.