Failure of CD4⁺ T cells to proliferate in response to antigenic stimulation is a characteristic of HIV infection. Analysis of the proliferation defect has been hampered by an inability to identify CD4⁺ cells with T cell receptor specificity for antigen. To focus only on cells that had been stimulated through the T cell receptor, CD4⁺ T cells were stimulated with an anti-Vβ3 Ab that activates approximately 3–5% of peripheral blood T cells. This approach revealed proliferation defects in cells from HIV-infected patients that were not appreciated using anti-CD3 Ab stimulation and provided the capacity to examine responses on a single cell basis. After anti-Vβ3 Ab stimulation, CD4⁺Vβ3⁺ cells from HIV-infected patients demonstrated defects in expression of cell cycle–associated proteins, D-type cyclins, and cyclin A. However, the expression of early activation markers, CD69 and CD25, was not significantly impaired in cells from most patients. Thus, CD4⁺ T cell proliferation failure in HIV disease is characterized by dysregulated activation that precludes cell cycle progression. This proliferation defect was most apparent in patients with diminished CD4⁺ T cell numbers and higher plasma HIV RNA levels. CD4⁺ T cell proliferation failure may be a key determinant of immune impairment in HIV disease.