Surveillance of cytomegalovirus (CMV) replication in transplant patients is crucial for the success of transplantation. To compare a CMV pp65 antigenemia (pp65Ag) and a quantitative real-time PCR targeting the CMV-UL111a (UL111aPCR), all whole blood samples taken between July 2008 and October 2009 were identified which had been analyzed prospectively by both assays in parallel. Discordant results were re-analyzed using a published CMV duplex PCR targeting regions UL55 and UL123exon4. Of 720 samples from 81 transplant patients, CMV replication was detected in 244 specimens (34%) by the UL111aPCR (median, 1,019 geq/ml), compared to 113 (16%) detected by the pp65Ag (median, 2/250,000 leukocytes). Concordant UL111aPCR/pp65Ag results were obtained in 561 (78%) samples, being positive in 99 (14%), and negative in 462 (64%). As a rule of thumb, 1 pp65Ag-positive cell per 250,000 leukocytes corresponded to 1,000 geq/ml CMV DNA of whole blood. Discordant results were found in 159 samples (22%), being UL111aPCR-positive/pp65Ag-negative in 145 (91%; median, 650 geq/ml), or UL111aPCR-negative/pp65Ag-positive in 14 (9%; median, 1/250,000 cells). Using the duplex PCR targeting the CMV UL55 and the UL123-exon4 genes, 131 of 139 (94%) discordant UL111aPCR-positives (median UL111aPCR, 639 geq/ml; median UL55PCR, 715 geq/ml; median UL123PCR, 1,103 geq/ml) were confirmed. Of 14 discordant pp65Ag-positives, duplex PCR was also negative in 8, and of low copy number in 6. Thus, CMV UL111aPCR provides more sensitive quantitation of CMV replication than pp65Ag, however, discordant results can occur at very low viral loads. J. Med. Virol. 83:2143–2150, 2011. © 2011 Wiley Periodicals, Inc.