The purpose of our work was to examine the most reliable laboratory diagnosis of fetal parvovirus B19 infection in hydropic fetuses by evaluating the most appropriate clinical sample and laboratory test. B19 DNA detection in fetal samples and serological signs of B19 infection in the respective mothers. Samples collected between January 2000 and July 2008. Microbiology, University of Bologna, Bologna, Italy. One hundred thirty-five fetal samples (58 fetal cord blood and 77 amniotic fluid samples) and 109 serum samples collected from 109 pregnant women. Validated and certified in situ hybridisation assay (ISH) and polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) were performed on fetal samples to detect B19 DNA. B19-specific antibodies were investigated in maternal serum samples by a commercial enzyme immunoassay. Parvovirus B19 DNA detection in fetal specimens was analysed in relation to maternal serological signs of infection. Parvovirus B19 DNA was detected in 22.41% of fetal cord blood and 36.36% of amniotic fluid samples. A statistically significant difference was found between DNA detection by ISH (23.70%) and PCR-ELISA (14.81%) (P = 0.004). Only 11.76% of fetuses with virological diagnosis of B19 infection were from women with serological signs of acute/recent B19 infection. Diagnosis of fetal parvovirus B19 infection cannot always rely on maternal serological investigations but rather on the virological analysis of fetal samples. Both fetal cord blood and amniotic fluid samples are suitable for diagnosis, but the detection of B19 DNA in the cells of amniotic fluid samples by ISH proved to be the most reliable diagnostic system.