The identification of a monoclonal antibody, AF3, which recognizes a single isoform of the cell surface protein CD44 and preferentially blocks binding of serotype 2 poliovirus to HeLa cells, suggested that CD44 might be an accessory molecule to Pvr, the cell receptor for poliovirus, and that it could play a role in the function of the poliovirus receptor site. We show here that only AF3 blocks binding of serotype 2 poliovirus to HeLa cells and, in contrast to a previously published report, that the anti-CD44 monoclonal antibodies A3D8 and IM7 are unable to block binding of poliovirus. To determine whether CD44 is involved in poliovirus infection, we analyzed the replication of all three serotypes of poliovirus in human neuroblastoma cells which lack or express CD44 and in mouse neuroblastoma cells which lack Pgp-1, the mouse homolog of human CD44, and which express Pvr. All three poliovirus serotypes replicate with normal kinetics and to normal levels in the absence or presence of CD44 or in the absence of Pgp-1. Furthermore, the binding affinity constants of all three poliovirus serotypes for Pvr are unaffected by the presence or absence of CD44 in the human neuroblastoma cell line. We conclude that CD44 and Pgp-1 are not required for poliovirus replication and are unlikely to be involved in poliovirus pathogenesis.