We report the isolation and characterization of HEp-2c cell clones obtained after two successive persistent poliovirus (PV) infections. Once cured, some of the cell clones displayed selective permissivity toward the wild-type Mahoney strain and partial resistance to particular mutants of this strain, including the Sabin 1 strain. Two cell clones, CI 4 and CI 10, were studied in greater detail. The cytopathic effects of Mahoney infection were comparable in the cell clones and in HEp-2c cells. The cytopathic effects of infection by Sabin 1 or Mahoney mutants were greatly delayed in CI 4 and CI 10. In the genomic region encoding the capsid proteins, determinants involved in the resistance of the cell clones to the Mahoney mutants were localized in the amino-terminal part of VP1 (amino acids 22 and 43), the B-C loop of VP1 (amino acids 94-102), and the loop of VP3 connecting its amino-terminal to beta strand B (amino acid 60). These genomic regions are thought to be involved in the early steps of viral infection. Virus adsorption was slower and less efficient on CI 10 cells than on parental HEp-2c cells. Virus adsorption was faster on CI 4 than on HEp-2c cells, and at least as efficient, but there was less receptor-induced structural modification of the capsid, a step that is required for decapsidation. Furthermore, infection of CI 4 by a Mahoney mutant in which the B-C loop of VP1 has been deleted was affected in the later steps of infection. These results indicate that, in cells cured of persistent PV infection, poliovirus multiplication was restricted at several stages and particularly at two steps of virus entry: adsorption and/or the uncoating transitions following adsorption onto the receptor.