The Centers for Disease Control and Prevention (CDC) recommends offering HIV testing to persons admitted to emergency departments (EDs). Whether by opt-in or opt-out, many EDs (including our own) have found a seroprevalence of 0.8-1.5% when rapid testing is offered. The true seropositivity rate is unknown. We performed a retrospective chart analysis upon all patients presenting to our ED over a 2-week period in the fall of 2007 who had serum drawn as a part of their emergency care. Demographics and clinical characteristics were linked via de-identified serum, which was sent for HIV testing. Nine hundred fifty nine patients had sera available for rapid HIV testing. One hundred twenty one (13%) samples were reactive via the OraQuick(®) test (OraSure Technologies, Bethlehem, PA), a point of care rapid antibody test. Due to concerns about the appropriateness of sera as substrate for the OraQuick(®) technology, reactive samples were retested via standard enzyme immunoassay (EIA)/Western blot. One hundred twelve analyzable samples were retested-38 were positive and 27 of these were from patients who reported a history of HIV infection. The rate of undiagnosed HIV infection was 1.2% (11/914 potentially analyzable samples). Of all patients with HIV in our ED, 29% of them were presumably unaware of their diagnosis. In conclusion, HIV seroprevalence in our urban ED is high, and a large fraction of the patients appears to be unaware of the infection.