Simian and human foamy virus (FV) DNA can be readily detected in peripheral blood leukocytes. However, it is unknown which leukocyte populations harbor the virus in vivo. We, therefore, analyzed blood samples from nine African green monkeys, four chimpanzees, and two humans for the presence of foamy virus proviral DNA in different FACS-purified leukocyte populations, using a highly sensitive nested polymerase chain reaction (PCR). The CD8+ lymphocytes were PCR positive in all 15 samples and the average viral burden was highest in this population. FV DNA was detected in 10 of 15 cell samples enriched for B lymphocytes, and 4 of 9 CD4+ lymphocyte, 3 of 13 CD14+ monocyte, and 4 of 13 polymorphonuclear leukocyte samples. A highly sensitive reverse transcriptase PCR was performed to detect viral transcripts in peripheral blood leukocytes. All samples were negative. In conclusion, lymphocytes, and especially CD8+ T lymphocytes, were found to be a major target for foamy virus in the peripheral blood, but viral gene expression was not detected.