In a previous study, we reported the isolation of a lambda gt11 cDNA clone (C3) for a virus message that mapped to the HindIII R fragment (human cytomegalovirus [AD169]). In this report, we further analyze transcription from this region of the genome. C3 was used to probe Northern (RNA) blots of RNA isolated from infected cells. Two abundant messages, 1.3 and 1.6 kilobases (kb) in size, were detected at 62 h postinfection (p.i.). Examination of different time points determined that the 1.6-kb mRNA accumulated in infected cells between 24 and 48 h p.i. and was classified as a late message. The 1.3-kb message was transcribed early in infection and was initially detected around 12 h p.i. Both transcripts were suppressed when infected cells were treated with inhibitors of DNA synthesis. Sequencing and S1 analysis identified the 5' ends of these two messages within 240 nucleotides of each other. Two CAAT-TATA motifs were found upstream of the 1.3- and 1.6-kb mRNA initiation sites, which suggested that the promoters were also closely associated. Antisera made to the fusion protein, synthesized from the C3 clone, detected a 25-kilodalton (kDa) virus protein found in infected cells and purified virions. Western blot (immunoblot) analysis of infected-cell proteins at various times after infection demonstrated that the accumulation of the 25-kDa protein coincided with the appearance of the 1.6-kb message. Therefore, we conclude that the 25-kDa virion protein is translated from the 1.6-kb message.