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Objective To investigate the effect of scaling up antiretroviral treatment (ART) on the working environment and motivation of health workers in South Africa; and to suggest strategies to minimize negative effects and maximise positive effects. Methods Exploratory interviews with health managers and senior clinical staff were used to identify locally relevant work environment indicators. A self -reported Likert scale questionnaire was administered to a randomly selected cohort of 269 health professionals at health facilities in KwaZulu Natal and Western Cape provinces of South Africa that included ART delivery sites. The cohort was disaggregated into ART and non-ART groups and differences between the two compared with Fisher’s exact test and the non-parametric Mann–Whitney U-test. Results The ART sub-cohort reported: (i) a lighter workload ( P = 0.013), (ii) higher level of staffing ( P = 0.010), (iii) lower sickness absence ( P = 0.032), (iv) higher overall job satisfaction ( P = 0.010), (v) poorer physical state of their work premises ( P = 0.003), and (vi) higher staff turnover ( P = 0.036). Conclusion Scale-up affects the work environment in ways that influence workers’ motivation both positively and negatively. A net negative balance is likely to drive staff out-migration, undermine the quality of care and compromise the capacity of the programme to achieve significant scale. As health workers are the most important element of the health system, a comprehensive and systematic understanding of scale-up impacts on their working conditions and motivation needs to be an integral part of any delivery strategy. [ABSTRACT FROM AUTHOR]