To estimate the 2009-2010 undiagnosed-HIV prevalence in the Paris metropolitan region, where half of France's new HIV cases are diagnosed annually, we used a direct method based on a large sample of emergency department patients unaware of their HIV status. The overall expected prevalence was 0.09% (95% confidence interval 0.04-0.13). Undiagnosed infections were exclusively found in high-risk groups. This prevalence is below the 0.1% threshold suggested by regulatory authorities for implementing universal screening.