The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) expresses IE86, IE72, IE55, and IE18 mRNA by differential splicing. Reverse transcription-PCR with IE72-specific primers generated an 0.65-kb cDNA from HCMV-infected fibroblast RNA, which does not correspond to any known MIE cDNA. Nucleotide sequencing revealed that the 0.65-kb cDNA is from exons 1, 2, and 3 and part of exon 4, indicating that it is derived from a novel alternatively spliced mRNA of the MIE gene. The cDNA encodes a 172-amino-acid polypeptide, termed IE19, which corresponds to an IE72 variant with an internal deletion from Val(86) to Pro(404) and appears as a band at 38 kDa on a sodium dodecyl sulfate-polyacrylamide gel. IE19 mRNA was expressed at a low level in the immediate-early, early, and late period of viral infection. IE19 was localized in nuclei, and a transient-expression assay revealed that IE19 enhances IE72-dependent activation of the HsOrc1 promoter, which is identified here as an IE72 target promoter. Another MIE protein, IE86, activated the same promoter but only weakly compared to IE72, and coexpression of IE19 did not alter the IE86-mediated transcriptional activation. In addition, IE19 did not enhance the IE72-dependent activation of the HCMV UL54 promoter. These results suggest that IE19 is a transcriptional coactivator that works with IE72.