Cell lines of primate origin carry receptors on their plasma membrane which are responsible for the specific binding of poliovirus. This paper describes the isolation and characterization of a monoclonal antibody reacting with the plasma membrane of HeLa cells. The antibody (D171) was selected for its protection of HeLa cells against the cytopathic effect of poliovirus type 1. This protection was found to extend to all three viral serotypes, while the replication of five other viruses in HeLa cells was not affected. The 125I-labelled purified antibody did not react with cell lines derived from pig, dog or rodents but bound specifically to all lines of human or primate origin. Immunoglobulin or Fab fragments of D171 prevented the binding of 35S-labelled poliovirus to HeLa cells. Conversely, nearly all binding sites of 125I-labelled D171 immunoglobulins or Fab fragments could be blocked after preincubation of HeLa cells with poliovirus. These results indicate that D171 recognizes the poliovirus receptor site on different susceptible cells and that practically all D171 binding sites are involved in the specific attachment of poliovirus to the plasma membrane. To determine whether the epitope recognized by D171 could be separated from the receptor for poliovirus, human-mouse cell hybrids were prepared and analysed. In all 40 clones tested, the susceptibility to poliovirus correlated with the binding of D171.