Background: It has been predicted that the UL31 gene originates from the positive strand of the human cytomegalovirus (HCMV) genome, whereas the UL30 and UL32 genes originate from the complementary strand. Except for the UL32 gene, the transcription of this gene region has not been investigated extensively. Methods: Northern blotting, cDNA library screening, RACE-PCR, and RT-PCR were used. Results: At least eight transcripts of the antisense orientation of UL31 were transcribed from the UL30-UL32 region during the late phase of HCMV infection. The 3' coterminus of these transcripts was located within the predicted UL30 gene. The longest 6.0-kb transcript was initiated upstream of the predicted UL32 gene. Other transcripts were derived from the predicted UL30 and UL31 gene region. Except for the previously predicted UL32 open reading frame (ORF), three novel ORFs, named UL31anti-1, UL31anti-2 and UL31anti-3, were located in the transcripts from the UL31anti-UL32 transcription unit. No transcription was found in UL31. Conclusion: A family of novel 3' coterminal transcripts was transcribed from the UL30-UL32 gene region.