Background: Shortages of health workers are obstacles to utilising global health initiative (GHI) funds effectively in Africa. This paper reports and analyses two countries’ health workforce responses during a period of large increases in GHI funds. Methods: Health facility record reviews were conducted in 52 facilities in Malawi and 39 facilities in Zambia in 2006/07 and 2008; quarterly totals from the last quarter of 2005 to the first quarter of 2008 inclusive in Malawi; and annual totals for 2004 to 2007 inclusive in Zambia. Topic-guided interviews were conducted with facility and district managers in both countries, and with health workers in Malawi. Results: Facility data confirm significant scale-up in HIV/AIDS service delivery in both countries. In Malawi, this was supported by a large increase in lower trained cadres and only a modest increase in clinical staff numbers. Routine outpatient workload fell in urban facilities, in rural health centres and in facilities not providing antiretroviral treatment (ART), while it increased at district hospitals and in facilities providing ART. In Zambia, total staff and clinical staff numbers stagnated between 2004 and 2007. In rural areas, outpatient workload, which was higher than at urban facilities, increased further. Key informants described the effects of increased workloads in both countries and attributed staff migration from public health facilities to non-government facilities in Zambia to PEPFAR. Conclusions: Malawi, which received large levels of GHI funding from only the Global Fund, managed to increase facility staff across all levels of the health system: urban, district and rural health facilities, supported by task-shifting to lower trained staff. The more complex GHI arena in Zambia, where both Global Fund and PEPFAR provided large levels of support, may have undermined a coordinated national workforce response to addressing health worker shortages, leading to a less effective response in rural areas.