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Countries with high HIV prevalence face the challenge of achieving high coverage of antiretroviral drug regimens interventions including for the prevention of mother-to-child transmission of HIV (PMTCT). In 2011, the World Health Organization and the Department of Foreign Affairs, Trade and Development, Canada, launched a joint implementation research (IR) initiative to increase access to effective PMTCT interventions. Here, we describe the process used for prioritizing PMTCT IR questions in Malawi, Nigeria, and Zimbabwe. Policy makers, district health workers, academics, implementing partners, and persons living with HIV were invited to 2-day workshops in each country. Between 42 and 70 representatives attended each workshop. Using the Child Health Nutrition Research Initiative process, stakeholder groups systematically identified programmatic barriers and formulated IR questions that addressed these challenges. IR questions were scored by individual participants according to 6 criteria: (1) answerable by research, (2) likely to reduce pediatric HIV infections, (3) addresses main barriers to scaling-up, (4) innovation and originality, (5) improves equity among underserved populations, and (6) likely value to policy makers. Highest scoring IR questions included health system approaches for integrating and decentralization services, ways of improving retention-in-care, bridging gaps between health facilities and communities, and increasing male partner involvement. The prioritized questions reflect the diversity of health care settings, competing health challenges and local and national context. The differing perspectives of policy makers, researchers, and implementers illustrate the value of inclusive research partnerships. The participatory Child Health Nutrition Research Initiative approach effectively set national PMTCT IR priorities, promoted country ownership, and strategically allocated research resources. [ABSTRACT FROM AUTHOR]