Background:The World Health Organization recommends that antiretroviral therapy (ART) programs in resource-limited settings develop third-line ART policies. South Africa developed a national third-line ART program for patients who have failed both first-line non-nucleoside reverse transcriptase inhibitor–based ART and second-line protease inhibitor (PI)-based ART. We report on this program.Methods:Third-line ART in South Africa is accessed through a national committee that assesses eligibility and makes individual regimen recommendations. Criteria for third-line include the following: ≥1 year on PI-based ART with virologic failure, despite adherence optimization, and genotypic antiretroviral resistance test showing PI resistance. We describe baseline characteristics and resistance patterns of this cohort and present longitudinal data on virological suppression rates.Results:Between August 2013 and July 2014, 144 patients were approved for third-line ART. Median age was 41 years [interquartile range (IQR): 19–47]; 60% were women (N = 85). Median CD4+ count and viral load were 172 (IQR: 128–351) and 14,759 (IQR: 314–90,378), respectively. About 2.8% started PI-based ART before 2004; 11.1% from 2004 to 2007; 31.3% from 2008 to 2011; and 6.3% from 2012 to 2014 (48.6% unknown start date). Of the 144 patients, 97% and 98% had resistance to lopinavir and atazanavir, respectively; 57% had resistance to darunavir. All were initiated on a regimen containing darunavir, with raltegravir in 101, and etravirine in 33. Among those with at least 1 viral load at least 6 months after third-line approval (n = 118), a large proportion (83%, n = 98) suppressed to <1000 copies per milliliter, and 79% (n = 93) to <400 copies per milliliter.Conclusion:A high proportion of third-line patients with follow-up viral loads are virologically suppressed.