The highly infectious phase of acute human immunodeficiency virus (HIV) infection, defined as the interval between the appearance of HIV RNA in plasma and the detection of HIV-1-specific antibodies, contributes disproportionately to HIV transmission. The current HIV diagnostic algorithm consists of a repeatedly reactive immunoassay (IA), followed by a supplemental test, such as the Western blot (WB) or indirect immunofluorescence assay (IFA). Because current laboratory IAs detect HIV infection earlier than supplemental tests, reactive IA results and negative supplemental test results very early in the course of HIV infection have been erroneously interpreted as negative. To address this problem, CDC has been evaluating a new HIV diagnostic algorithm. This report describes two evaluations of this algorithm. An HIV screening program at a Phoenix, Arizona emergency department (ED) identified 37 undiagnosed HIV infections during July 2011-February 2013. Of these, 12 (32.4%) were acute HIV infections. An ongoing HIV testing study in three sites identified 99 cases with reactive IA and negative supplemental test results; 55 (55.6%) had acute HIV infection. CDC and many health departments recognize that confirmatory supplemental tests can give false-negative results early in the course of HIV infection. This problem can be resolved by testing for HIV RNA after a reactive IA result and negative supplemental test result.