Fourteen immunocompromised patients were examined for viremia, pp65 and p72 antigenemia, and presence of viral DNA in leukocyte fractions of polymorphonuclear leukocytes (PMNL), monocytes/macrophages (M/M), and B and T lymphocytes after purification by fluorescence-activated cell sorting. Nearly all PMNL and M/M fractions were positive for DNA and pp65 antigenemia, while p72 antigenemia was detected in 73% and 62%, respectively. The virus isolation rate was 45% from PMNL and 17% from M/M. T lymphocytes were positive for DNA in 50% of cases and for pp65 and p72 antigenemia in only 11%, while B lymphocytes were DNA-positive in 43% of samples and consistently negative for antigenemia; neither T nor B lymphocytes had virus isolated. Immediate-early (IE)1 RNA was present in 23 (85.2%) of 27 dextran-enriched DNA-positive p72-positive PMNL samples and, in sequential PMNL samples from two heart-transplanted patients, was detected during peak infection in association with p72. Thus, PMNL and M/M are the subpopulations primarily involved in HCMV infection; PMNL may undergo IE replicative events and are not merely passive carriers of phagocytized viral material.