Erythroid lineage cells derived from fetal liver were demonstrated to be target cells for human parvovirus B19 infection. B19 virus antigen-positive serum was inoculated into primary cultures containing erythroid lineage cells enriched from fetal liver. The B19 virus antigen was detected on about 5% of cells in the culture by immunofluorescence staining, and the stained cells were identified as erythroid lineage cells by double staining with anti-B19 virus-positive serum and anti-erythroid lineage monoclonal antibody. The immunofluorescence staining study also revealed that the B19 virus antigen localized in the nucleus and the periphery of cytoplasm. We also detected B19 virus DNA, which was generated by replication in the infected cells, not only in the cells but also in the culture supernatants, in which the amount of B19 DNA increased depending on the period of culture, indicating that the cells infected with B19 virus produced B19 virus and released it into the medium. The ability of B19 virus released into the medium to infect fetal erythroid lineage cells was demonstrated quantitatively. Because of the absence of any cytopathic effect of B19 virus during culture periods of at least 15 days, this culture system should be useful in the study of B19 virus replication and in vitro generation of B19 virus. In addition, the present study may contribute to a better understanding of the pathogenesis of hydrops fetalis, which is probably associated with B19 virus infection during pregnancy.