The enzyme-linked immunospot (ELISPOT) assay was adapted to detect and enumerate HIV-1-producing cells at the single cell level. With CEM cells or peripheral blood mononuclear cells (PBMC) infected in vitro with HIV-1, the ELISPOT assay detected cells that produced HIV-1 antigens and showed that between 5.4% and 9.5% of the p24 antigen-positive CEM cells and 11.1% to 23.6% of the p24 antigen-positive PBMC were productively infected. In HIV-1-infected patients in early stage of the disease and without antiretroviral therapy, up to 4.54 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in peripheral blood and up to 277.75 HIV-1-producing cells per 10(6) CD4+ T lymphocytes were detected in splenic lymphoid tissue. Our results indicate that the ELISPOT assay could represent a new tool to study HIV-1 replication in vivo.