In this study, we demonstrated that antisense transcripts of human cytomegalovirus (HCMV) UL123, UL21.5 and cellular GAPDH genes were present in highly purified virions. These virion RNAs were delivered into the host cells upon infection, and de novo synthesized ones appeared in the infected cell at the immediate early stage. Although the sequence of UL123 antisense transcripts in virions is uncertain, we found that these transcripts in Towne-infected human fibroblasts had novel transcriptional start sites (TSSs) with various 5'-terminal deletions of open reading frame (ORF) 59. These findings not only provide new insight into the composition of HCMV virions but also reveal a possible viral strategy for initiating latent infection and switching between latent and productive infections.