We report the first case, to our knowledge, of antibody-negative human immunodeficiency virus type 1 (HIV-1) subtype C infection, which was identified during screening for acute HIV-1 infection in Botswana. Results of tests for HIV-1 antibodies were consistently negative, including rapid and regular enzyme-linked immunosorbent assay and Western blot. The nonrecombinant HIV-1 subtype C infection was confirmed by viral genotyping within the gag, pol, and env genes. The period between referral of the patient in a clinically stable condition and AIDS-related death was approximately 3 months. The reported case indicates the importance of studying the prevalence of seronegative HIV-1 infection in southern Africa, where subtype C predominates.