Background The Human Immunodeficiency Virus (HIV) causes the Acquired Immune Deficiency Syndrome (AIDS). The AIDS disease is a recognised global epidemic affecting men, women and children. In sub-Saharan Africa, women and children are particularly vulnerable with about 61% of the infections occurring in women. Of note is that about 90% of the paediatrics infection is through Mother – To – Child Transmission (MTCT). This makes elimination of MTCT highly imperative. In Nigeria, antenatal attendees are offered routine HIV counselling and testing on the 1st visit with optout option. This is done irrespective of the gestational age at contact. This appears commendable but, considering the national HIV prevalence of 4.2%, our large population of > 140 million, the long period of seroconversion of the virus and the fact that pregnant women continue to indulge in activities that put them at risk of new infections; a single screening test on contact may not be sufficient to detect all maternal infections. Aim and objectives We re-tested pregnant women later in pregnancy and determined the seroprevalence of HIV amongst antenatal attendees who tested negative in the 1st half of pregnancy. We also determined the factors associated with repeat sero-positive result as well as the acceptability of the repeat counselling and testing to the women. Materials and methods This is a prospective cross sectional study conducted in Nnamdi Azikiwe University Teaching Hospital (NAUTH) Nnewi between November 2010 and February 2011. The antenatal attendees that gave consent for the re-testing had their venous blood collected via standard venipuncture techniques. The rapid test kits: Determine, Stat Pak and Unigold were used for detection/diagnosis of HIV using the serial test algorithm II after counselling. Semi structured questionnaire was used to collect data on the sociodemographic characteristics and factors that may contribute to acquisition of the infection. Descriptive analysis of the result was done using the Statistical Package for Social Sciences Version 16 (SPSS, Chicago, IL, USA). P<0.05 was considered significant (providing 95% confidence interval).