The prevalence of human immunodeficiency virus antibodies in mothers and their neonates was determined through unlinked anonymous HIV testing at delivery. Two hundred and forty-six apparently healthy volunteer anonymous parturients at the University of Benin Teaching Hospital, Nigeria, and their neonates were recruited for the study. Blood samples were collected from the mothers and cord blood from their neonates at delivery. The blood samples were coded and assayed for HIV antibodies using ImmunoComb HIV 1 and 2 bispot test and ImmunoComb II HIV-1 and 2 CombFirm, both from PBS Orgenics, France. The results for maternal and neonatal blood sample pairs were matched. The maternal seroprevalence for HIV antibodies was 2.4% (6/246) while only two infants had HIV antibodies. The mother-to-child transmission of HIV antibodies was 33.3%. The study highlights the increasing HIV infection among pregnant women in this hospital. The risk of vertical transmission is therefore high. Universal antenatal HIV testing with an opt-out system is suggested. The need for our maternity centres and special care baby units to establish management protocols and anti-retroviral therapy for HIV infected women and their neonates is recommended.