Humans tailor virus-specific immune responses through modulated expression of 12 different interferon (IFN)-alpha subtypes. However, exacerbated expression of certain IFN-alpha subtypes causes immunopathology in the context of autoimmune conditions and chronic viral infections. We showed that progression to AIDS is associated with elevated expression of IFN-alpha in unstimulated peripheral blood mononuclear cells. Here, we sought to determine whether distinct IFN-alpha subtypes are involved in this phenomenon. We used quantitative RT-PCR to assess expression levels of 12 IFN-alpha subtypes in peripheral blood mononuclear cells from normal donors and HIV-1 patients at CDC stage A and stage C of the disease. Three patterns of IFN-alpha subtype expression emerged. First, IFN-alpha2 and IFN-alpha6 mRNA levels were elevated in both patient groups. Second, IFN-alpha1/13, IFN-alpha8, IFN-alpha14, IFN-alpha16, IFN-alpha17, and IFN-alpha21 were upregulated in stage C but not stage A patients. Third, expression levels of IFN-alpha4, IFN-alpha5, IFN-alpha7, and IFN-alpha10 did not change among the three groups of volunteers. Among all other subtypes, IFN-alpha2 was preferentially upregulated, showing >60-fold higher levels in stage A and >400-fold in stage C patients compared with controls, which correlated with declining CD4 counts. Our results demonstrate that distinct IFN-alpha subtypes are sequentially activated during HIV-1 infection, which may be predictive of disease progression.