In eastern and southern Africa, much is unknown about implementation of nurse-initiated and managed antiretroviral therapy (NIMART). The purpose of this study was to identify perceived barriers and facilitators of NIMART for the prevention of mother-to-child transmission and pediatric HIV services in high-volume, high HIV-burden health facilities across this region. A total of 211 nurses, midwives, and nurse midwives and 62 supervisors from 30 health facilities in 11 countries participated in this mixed-methods evaluation. The findings show that although nurses, midwives, and nurse midwives clearly had the authority to provide NIMART services, they did not necessarily feel that they were well prepared and supported to do so. Deficits in supportive supervision and clinical mentorship were viewed as substantial challenges to effective provision of NIMART for the prevention of mother-to-child transmission and pediatric HIV services-particularly with respect to pediatric HIV services. Health facilities have important opportunities to advance NIMART practice through strengthening these aspects of in-service support.