OBJECTIVES: This prospective study assessed the costs and cost-effectiveness of routine HIV testing in non traditional settings using Oral fluid-based HIV testing and a fourth-generation assay. METHODS: HIV tests were offered to patients aged 16-65 over 3 months in 4 settings: Emergency Department, Acute Care Unit, Dermatology Outpatients and Primary Care in London.  We assessed and compared the costs and the cost-effectiveness ratio of screening in terms of costs for newly diagnosed HIV-infected patients using the real data derived from each setting.   Additionally, the Survey of Prevalent HIV Infections Diagnosed (SOPHID) was used to estimate the number of undiagnosed individuals attending each setting over one year. A sensitivity analysis was run using the SOPHID data to simulate the costs and cost-effectiveness of the HIV screening in different scenarios, changing the prevalence and the compliance rates. RESULTS: HIV testing in non traditional settings cost £19,056.31 per newly diagnosed patient. Using the SOPHID data and assuming the same compliance rate, the cost for a newly diagnosed patient is £4,460.59. In the best scenario, assuming 100% compliance, the cost will decrease to £2,940 per patient. These figures do not take into account the additional cost savings that may result from earlier diagnosis of HIV-infected individuals. CONCLUSIONS: The results of the study are really encouraging and suggest that a screening programme in a high prevalence area could identify HIV-infected patients at a very low cost.  Earlier diagnosis of HIV infection may subsequently have further cost benefits in terms of aversion of incident infections and early treatment of infected individuals.