The interaction of poliovirus with its cellular binding sites was characterized by using a receptor-excess silicon oil partition assay. Poliovirus type 1 Mahoney [PV1(M)] binding to HeLa cells fits a theoretical simple bimolecular noncooperative binding curve with an equilibrium dissociation constant (Kd) of 4.3 × 107 cells· ml-1 at 4°, or 2.1 × 10-10M, assuming 3000 virus binding sites/cell. The association rate of complex formation was measured to be 3.6 × 10-9 ml·cell-1·min-1 (7.2 × 108M-1·min-1) and the dissociation rate calculated to be 1.5 × 10-1 min-1, giving the complex a half-life of 4.5 min. The equilibrium dissociation constant, association rate, and dissociation rate were also measured for the binding of the attenuated poliovirus type 3 Sabin strain [PV3(S)] to HeLa cells. PV3(S) bound HeLa cells with a Kd of 3.3 × 107 cells·ml-1 (1.6 × 10-10M), an association rate of 4.1 × 10-9 ml·cell-1 min-1 (8.2 × 108M-1 ·min-1), and a dissociation rate calculated to be 1.4 × 10-1 min-1, giving the complex a half-life of 5.1 min. Thus the virulent and avirulent strains of poliovirus bind HeLa cells with nearly identical binding constants and rate constants. Equilibrium binding constants for PV1(M) to various other cell types varied from a high affinity of 4.1 × 106 cells·ml-1 for JA-1 cells to a low affinity of 7 × 107 cells·ml-1 for NGP cells. Comparison of the amount of immunoprecipitated poliovirus receptor suggests that the weak virus binding of NGP cells may be due to a limited number of receptors expressed on the cell surface of these cells. © 1994 Academic Press. All rights reserved.