Acute infection with human immunodeficiency virus (HIV) is often accompanied by a flu-like illness, and early identification and treatment may help control the infection and prevent transmission. We enrolled patients who presented to an urban urgent care center with any symptoms of a viral illness and any recent potential risk for HIV infection, and we tested them for acute HIV infection using enzyme-linked immunosorbent and RNA assays. Of 499 patients enrolled over a 1-year period, acute HIV infection was diagnosed in 5 (1.0%; 95% confidence interval [CI], 0.1%–1.9%), and chronic HIV infection was diagnosed in 6 (1.2%; 95% CI, 0.2%–2.2%). There were no false-positive results of the RNA assay. No signs or symptoms reliably distinguished patients with acute HIV infection from those who were HIV uninfected. Given the importance of this diagnosis, testing for acute HIV infection using RNA and antibody assays should be offered to all patients in similar settings with viral symptoms and any risk factors for HIV infection.