The rapid advances in research on antisense transcripts are gradually changing our understanding of the expression of the Herpesviridae genome. In this study, the transcripts of the human cytomegalovirus (HCMV) UL83 antisense strand were investigated in three clinical isolates. Three cDNA clones containing sequences with an antisense orientation to the UL83 gene were identified in a late HCMV cDNA library. The UL83 antisense transcripts (UL83asts) were then shown to be transcribed only in the late infection phase of the three clinical HCMV strains, using rapid amplification of cDNA ends (RACE) and northern blotting. These UL83asts were identical at their 3 termini but different at 5 ends. Two open reading frames were predicted in the UL83asts. J. Med. Virol. 86:2033-2041, 2014. (c) 2014 Wiley Periodicals, Inc.