Objective: To determine the prevalence of HIV infection and the extent of disease progression based on CD4 count in a public health system workforce in southern Africa. Design: Cross-sectional voluntary, anonymous, unlinked survey including an oral fluid or blood sample and a brief demographic questionnaire. Setting: Two public hospitals in Gauteng, South Africa. Subjects: All 2 032 professional and support staff employed by the two hospitals. Outcome Measures: HIV prevalence and CD4 cell count distribution. Results: Overall prevalence of HIV was 11.5%. By occupation, prevalence was highest among student nurses (13.8%) and nurses (13.7%). The highest prevalence by age was in the 25 – 34-year group (15.9%). Nineteen per cent of HIV-positive participants who provided blood samples had CD4 counts less than or equal to 200 cells/ microl 28% had counts 201 – 350 cells/ microl, 18% had counts 351 – 500 cells/ microl, and 35% had counts above 500 cells/ microl. Conclusions: One out of 7 nurses and nursing students in this public sector workforce was HIV-positive. A high proportion of health care workers had CD4 counts below 350 cells/ microl, and many were already eligible for antiretroviral therapy under South African treatment guidelines. Given the short supply of nurses in South Africa, knowledge of prevalence in this workforce and provision of effective AIDS treatment are crucial for meeting future staffing needs.