Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.