The human herpesvirus-8 (HHV-8) locus encoding ORFs 40/41 is a candidate homologue for the Epstein-Barr virus BBLF 2/3 gene, which encodes the putative primase-associated factor. Northern blot data revealed that two transcripts originated from the HHV-8 ORF 40/41 region. The sizes of these transcripts (2.2 and 0.7 kb) suggested that one transcript was the result of a spliced form of ORFs 40 and 41 and the second transcript originated from a region within ORF 41. cDNA sequence and 5' RACE analysis revealed the removal of an intron between ORFs 40 and 41 and a transcriptional start site 82 nt upstream of ORF 40. The start of transcription for the smaller transcript was mapped to within ORF 41. Regions upstream of the transcriptional start sites were subcloned into a luciferase reporter vector, and transient luciferase assays indicated that distinct promoters drive the expression of each transcript.