We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.