Macrophages are often considered as a reservoir of latent infection in HIV+ patients, and their infection may indeed be very important functionally. However, some quantitative studies did not find high infection frequencies in peripheral blood monocytes. Since lymphoid organs are the major site of infection, macrophage infection was tested in spleens removed from HIV+ patients for treatment of different syndromes. Ten replicates of limiting dilutions from different cell populations were submitted to a nested PCR specific to conserved regions of HIV1 env DNA. On an average, 1/2,300 adherent cells carried HIV1 DNA (n = 7; range: 1/55,000 to 1/660). These adherent cells, obtained after two days of culture, comprised the whole macrophage population, with no biases introduced by surface molecule selection, but were not pure (41-78% macrophages). Only 1/37,000 CD14+ monocyte/macrophages were positive (n = 6; range: 1/130,000 to 1/22,000). Therefore, the infection frequency of the isolated splenic monocytes/macrophages from these patients could be estimated at between 1/37,000 and 1/2,300. In contrast, 1/60 CD4+ T lymphocytes were positive (n = 7; range: 1/190 to 1/17). Within the experimental limits, such as cell isolation, required for accurate quantification, this study in the spleen indicates, as have other studies on peripheral blood, that macrophages do not quantitatively constitute an important reservoir of HIV when compared to CD4+ T lymphocytes.