The proportion and significance of HIV-1 infection of CD8+ T-cells was examined in a patient cohort of HIV-1 seropositive (n=28) and seronegative individuals (n=4). It was hypothesized that irrespective of the clinical status of the patients, productively HIV-1 infected CD8+ T-cells would be found and these cells would contribute to the plasma viral load in vivo. Flow cytometric analysis using fluorochrome-conjugated antibodies, RT-PCR analysis using HIV-1(pol) specific primers, and quantification of HIV-1 viral transcripts by ex vivo culture of isolated CD8+ T-cells were employed. In 22 of the 28 patient samples analyzed, a significantly higher proportion of cells with expression of CD8+HIV-1(gag)+ than of CD4+HIV-1(gag)+ T-cells was observed (36.9% +/- 10.0% vs 26.4% +/- 13.1% respectively, p< 0.01). No correlation was observed between absolute CD4 counts, CD8 counts, plasma viral load and CD8+ T cell infection. RT-PCR analysis indicated the presence of HIV-1 transcripts in the ex vivo isolated CD8+ T-cell population. Ex vivo isolated CD8+ T-cells demonstrated productive infection over time. We conclude, with three lines of evidence detecting and measuring HIV-1 infection of CD8+ T-lymphocytes, that this cellular target and reservoir may be central to HIV-1 pathogenesis.