Diagnosis of acute HIV is done by patient history and examination and testing of RNA, proviral DNA, and serology using fourth-generation antigen/antibody detection assays. We describe an HIV-1 primary infection with a second diagnostic window of 18 to 34 days on a fourth-generation immunoassay, which would have been missed using some current algorithms. Caution must be exercised when fourth-generation HIV-1 immunoassays are interpreted in isolation, and additional testing should be considered depending on patient risk assessment.