We previously identified a 101-kDa apparent molecular mass polypeptide (101K) as the major immunoreactive virion protein of human herpesvirus 6 variant B strain Z29 [HHV-6B(Z29)] and found that the human immune response to this protein is HHV-6-specific (Yamamoto, M., Black, J. B., Stewart, J. A., Lopez, C., and Pellett, P. E., 1990, J. Clin. Microbiol. 28, 1957-1962). We report here the identification and characterization of the gene encoding 101K. We found 81% amino acid identity between an HHV-6B(Z29) open reading frame (ORF) and its homolog in HHV-6A strain U1102 [HHV-6A(U1102)]. The product of this gene was identified as 101K on the basis of both the reactivity of a 101K-specific monoclonal antibody (MAb C3108-103) with a bacterially expressed portion of the gene and the reactivity of polyclonal rabbit antibodies raised against the bacterially expressed protein with 101K expressed by HHV-6B(Z29)-infected cells. MAb C3108-103 reacted with eight of eight variant B isolates and none of six variant A isolates, indicating that it is a variant-specific MAb. The MAb reactivity was mapped to an eight-amino-acid segment of 101K. HHV-6A(U1102) differs from HHV-6B(Z29) by two amino acids in this region; substitution mapping with synthetic oligopeptides mapped the variant B specificity to Asp723, this explaining the failure of the MAb to react with variant A proteins. A set of transcripts appropriately sized for expression of 101K was identified and precisely mapped. The transcripts originated down-stream from either of two TATA boxes located 139 bp apart in the region 5' to the 101K ORF, with one 5'-species being much more abundant. Two independent polyadenylation sites were identified; the canonical polyadenylation signal located 3' to the 101K ORF was used much more frequently than was the atypical polyadenylation signal located within the 101K ORF. These results suggest a complex regulatory mechanism for this gene.