The most abundantly transcribed HCMV gene (β2.7) encodes a 2.7 kb polyadenylated RNA. Although the laboratory-adapted HCMV strains AD 169 and Towne possess two copies of the β2.7 gene within an expanded b sequence element, the low passage strain Toledo and all clinical isolates analysed contain only a single copy located within the UL region. A β2.7 deletion mutant constructed based on a strain Toledo background was shown to replicate with kinetics comparable to those of the parental virus; the β2.7 gene is therefore not essential for virus replication in vitro. Sequencing the β2.7 gene from HCMV clinical isolates and the Toledo strain reveals that although the overall gene sequence is highly conserved (> 99%), the RL4 frame originally assigned in strain AD169 was disrupted in each of these viruses. Consequently, the β2.7 transcript does not encode any obvious translation product and thus may not function as an mRNA.