The abnormalities of B lymphocytes in human immunodeficiency virus (HIV) infection usually have been attributed to secondary consequences of disturbed immunoregulation. However, recent work has revealed avid binding of HIV gp120 to a conserved immunoglobulin motif unique to the VH3 gene family and the selective gp120-induced activation of VH3 B cells in vitro. This cross-sectional clinical study tests whether HIV infection is associated with a selective response of VH3 B cells in vivo. Levels of blood B cells and serum immunoglobulins (Ig) expressing the D12 idiotope of the VH3 gene family were measured in subjects of the Multicenter AIDS Cohort Study grouped according to clinical stages of HIV infection. A 3-fold elevation in D12 B cells was observed in HIV seropositive versus seronegative groups. This was selective for the D12 population, since total B cell numbers were identical in the two groups. The levels of D12 B cells and CD4 T cells were significantly correlated, and D12 B cells were markedly reduced in the AIDS group (4- and 10-fold, compared to the seronegative and seropositive groups). Analogous differences were also seen in the levels of serum anti-gp120 D12 Ig. This change in VH3 B cells among groups of HIV-infected individuals provides further evidence identifying gp120 as a VH3 B cell superantigen.