In 2005 we implemented an emergency department HIV testing program that emphasized screening by nurses but also allowed for clinician diagnostic testing. We noted that clinicians often ordered tests that proved to be positive on patients who had been missed by screening, while others who tested positive had made previous visits when screening was available, but were not tested. The study objective was to quantify missed screening opportunities and assess the extent to which diagnostic testing contributes to the detection of HIV infection. Triage nurses were to offer screening to medically stable patients 12 years of age or older. Clinicians could order diagnostic testing in patients with signs and symptoms concerning for HIV. Nurses performed rapid HIV tests on oral fluid specimens. Charts of all patients testing positive between April 1, 2005 and November 31, 2006 were reviewed. The 2006 annual census was 75,000 visits with 47% of patients black, 32% Hispanic, 44% female, and 98% 12 years of age or older. Ninety-five patients tested HIV positive; 66 (69.5%) were diagnosed on their first visit but 29 (30.5%) made a total of 59 visits (range, 1-8) before testing positive. Patients were screening eligible during 54 (91.5%) of these 59 visits but screening was not offered during 34 (63.0%) of them, representing missed screening opportunities. On the day of diagnosis, 80 (84.2%) of the 95 patients were screening eligible but 20 (25.0%) of them were not offered screening, representing missed screening opportunities. Diagnostic testing identified HIV in 44 patients; 15 were screening ineligible, 20 were not offered screening, and 9 declined screening. Missed opportunities for earlier diagnosis occurred frequently despite an HIV screening program. Clinician diagnostic testing was an important adjunct to screening.