This study investigated the cell types infected with HIV in peripheral blood, and the relation of virus load in different cell subsets with disease progression. Two magnetic cell separation techniques were used to purify cell subsets from 34 HIV seropositive individuals at different stages of disease progression. HIV-1 DNA was detected in CD4 and CD8 lymphocytes, monocytes, natural killer cells and dendritic cells. The contribution to total proviral load by different subsets of peripheral blood mononuclear cells (PBMCS) was estimated by quantitative PCR combined with measurements of their relative frequency in peripheral blood. This analysis revealed that CD8 T lymphocytes are a major reservoir of HIV within the peripheral blood of individuals with AIDS. Further independent evidence for in vivo infection of CD8 lymphocytes was obtained by sequence comparisons of the V3 region of the envelope gene. Numerous sequences were obtained from each purified cell subset and initial sequence comparisons indicate that different virus populations may be present in CD4 and CD8 lymphocytes. The functional significance of this will remain unclear until such variants can be characterised virologically. An PCR based method was developed to detect spliced HIV mRNA transcripts whose presence within cells indicates active viral replication, and differentiates these from cells containing an inactive provirus. High levels of mRNA expression were detected in CD4 lymphocytes, CD8 lymphocytes and in one case in monocytes. This evidence indicates that mature CD8 lymphocytes can be actively infected with HIV-1 in vivo even although they do not express CD4.