In situ hybridization was used to detect human cytomegalovirus (HCMV) in the peripheral blood mononuclear cells of some naturally infected (seropositive) individuals. A subpopulation of cells hybridized specifically to a portion of the HCMV genome that is heavily transcribed during the immediate-early period of infection. The hybridization signal was markedly reduced by base hydrolysis and ribonuclease, and therefore the probe appears to be detecting viral RNA. A fluorescence-activated cell sorter was used to select lymphocytes bearing the OKT4 and OKT8 markers. Hybridization with the HCMV probe revealed a higher proportion of positive cells in the OKT4 than in the OKT8 subset. This observation specifically identifies lymphocytes as a cell population involved in natural HCMV infection and suggests that lymphocytes may be a reservoir for maintaining infection and may also serve as a vehicle for its spread by blood transfusion.