Leukocyte populations were purified by fluorescence-activated cell sorter and analyzed for human cytomegalovirus (HCMV) DNA and mRNA using polymerase chain reaction (PCR) and cDNA PCR, respectively. Twenty-two blood samples from 17 patients with active HCMV infection were studied. HCMV DNA was detected most frequently in granulocytes and monocytes but was also found in CD4+, CD8+, and CD19+ lymphocytes. Viral immediate early and late mRNA was found in ficoll-enriched mononuclear cells and polymorphonuclear leukocytes. In 11 samples from 9 patients with active HCMV infection, immediate early mRNA was detected in 8 lymphocyte, 6 monocyte, and 8 granulocyte fractions. Late mRNA was detected in 7 lymphocyte, 7 monocyte, and 9 granulocyte fractions. The presence of viral late mRNA provides strong evidence for infection of these leukocyte populations with HCMV in vivo.