BACKGROUND: An incorrect definition of immune status to human cytomegalovirus (HCMV) can lead to incorrect management of pregnant women. OBJECTIVES: Aims of the study were: i) to describe 10 cases of unconfirmed HCMV IgG-seroconversion in pregnancy; ii) to develop a panel of confirmatory tests to define HCMV serostatus; iii) to investigate the frequency of false IgG-positive results in pregnant women screened with the LIAISON(®)CMVIgGII automated assay. STUDY DESIGN: Blood samples from 10 pregnant women referred for HCMV IgG-seroconversion were examined to confirm/exclude a primary infection. In addition, samples were tested for HCMV IgG by immunoblotting, neutralization assay, and ELISA against gB, gH/gL/pUL128L and gH/gL/gO recombinant glycoproteins. LIAISON(®)CMVIgGII results obtained on 1158 pregnant women were reviewed and samples with low IgG titers were further investigated. RESULTS: A primary infection was excluded in the 10 women referred for HCMV IgG seroconversion. None of them was confirmed to be IgG-seropositive. Of the 1158 women prenatally screened by LIAISON(®)CMVIgGII, 678 (59%) were IgG-positive and, of these, 40 (5.9%) showed low levels of IgG (14-50 U/mL). Thirty-three women with low IgG-positivity were further tested by confirmatory tests and 11 (33.3%) were found to be non reactive to HCMV. CONCLUSIONS: At least 1.6% (11/678) women who tested positive with LIAISON(®)CMVIgGII were found to be seronegative when tested with confirmatory tests. These women should be informed to reduce the risk of a primary HCMV infection. Furthermore, should a congenital infection occur in any of these women, a maternal non-primary infection could be erroneously diagnosed.