Replication of the pathogenic human parvovirus B19 is restricted to erythroid progenitor cells. Although blood group P antigen has been reported to be the cell surface receptor for parvovirus B19, a number of nonerythroid cells, which express P antigen, are not permissive for parvovirus B19 infection. We have documented that P antigen is necessary for parvovirus B19 binding but not sufficient for virus entry into cells. To test whether parvovirus B19 utilizes a cell surface coreceptor for entry, we used human erythroleukemia cells (K562), which allow parvovirus B19 binding but not entry. We report here that upon treatment with phorbol esters, K562 cells become adherent and permissive for parvovirus B19 entry, which is mediated by alpha 5 beta 1 integrins, but only in their high-affinity conformation. Mature human red blood cells (RBCs), which express high levels of P antigen, but not alpha 5 beta 1 integrins, bind parvovirus B19 but do not allow viral entry. In contrast, primary human erythroid progenitor cells express high levels of both P antigen and alpha 5 beta 1 integrins and allow beta1 integrin-mediated entry of parvovirus B19. Thus, in a natural course of infection, RBCs are likely exploited for a highly efficient systemic dissemination of parvovirus B19.