OBJECTIVE: To estimate the incremental societal costs and effectiveness of a second human immunodeficiency virus (HIV) antibody test during the third trimester of pregnancy compared with no second test. METHODS: We used a decision tree in this cost-effectiveness analysis to model outcomes among pregnant women in high-risk communities and nationwide who received an initial, negative HIV antibody test during the first trimester. The main outcome measure was discounted costs per year of infant life saved. RESULTS: In high-risk communities with estimated HIV incidence of 6.2 per 1000 person-years, a second HIV test compared with no second test would detect 192 infections in women, prevent approximately 37 infant infections, and save 655 infant life-years per 100,000 women tested. Net savings would be $5.2 million. Applied to an estimated national incidence of .17 per 1000 person-years, a second test would detect 5.3 infections in women, prevent 1.3 infant infections, and save 23.3 infant life-years per 100,000 women tested. Net costs would be $1.06 million, or $45,708 for each year of infant life saved. A second test would result in net savings in populations with HIV incidence of 1.2 per 1000 person-years or higher. CONCLUSION: Health care providers serving women in communities with an HIV incidence of 1 per 1000 person-years or higher should strongly consider implementing a second voluntary universal HIV test during the third trimester. Providers serving lower-risk communities should pilot second testing to assess community-specific costs. (C) 2003 by The American College of Obstetricians and Gynecologists.