The in vivo interaction of human cytomegalovirus (HCMV) with leukocytes from a group of immunosuppressed patients was studied using specific subgenomic DNA probes and in situ cytohybridization. Study subjects had no recognized disease due to HCMV or had HCMV retinitis or colitis. Viral nucleic acid was detected in lymphocytes, monocytes, and polymorphonuclear leukocytes (PMNL). PMNL were consistently hybridization-positive for HCMV RNA and DNA in patients with HCMV viremia. Monocytes were occasionally positive by hybridization and lymphocytes were rarely positive. These findings demonstrate that the HCMV genome is expressed in PMNL of viremic patients and that PMNL may play an important role in virus dissemination.