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A decision analysis model, from a health care system perspective, was used to assess the cost-effectiveness of HIV rescreening during late pregnancy to prevent perinatal HIV transmission in South Africa, a country with high HIV prevalence and incidence among pregnant women. Because new HIV prenatal prophylactic and pediatric antiretroviral therapy (ART) regimens are becoming more widely available, the study was carried out with different combinations of the two. With an estimated HIV incidence during pregnancy of 2.3 per 100 person-years, HIV rescreening would prevent additional infant infections and result in net savings when zidovudine plus single-dose nevirapine or single-dose nevirapine is used for perinatal HIV prevention, and ART was available to treat perinatally HIV-infected children. The cost savings were robust over a wide range of parameter values when ART was available to treat perinatally HIV-infected children but were more sensitive to variations around the baseline when ART was not available. The minimum time interval between the initial and repeat screens would be from 3 to 18 weeks, depending on prophylactic and treatment regimens, for HIV rescreening to be cost saving. Overall, HIV rescreening late in pregnancy in high-prevalence, resource-limited settings such as South Africa would be a cost-effective strategy for reducing mother-to-child transmission.