The genomic region of human cytomegalovirus (HCMV) encoding the major immediate-early (IE) proteins was cloned as overlapping fragments into a prokaryotic expression vector. Three recombinant polypeptides were used as antigens to generate monoclonal antibodies specifically reactive with the proteins encoded by IE region 1 and IE region 2. At least 10 different antigenic regions were identified on the IE proteins of HCMV. One monoclonal generated against an IE-2 polypeptide of 156 amino acids (termed SMX) was found to react with a viral pentapeptide, which was also a constituent of the β-chain of human HLA-DR. This peptide was contained in the 40-kDa (p40) protein encoded by IE region 2. This protein appeared to be an abundant product of the major IE region in infected cells at late times after infection. The putative translation initiation site for p40 was mapped to position 3348 of the IE2 sequence using monoclonal antibody SMX. It is therefore proposed that p40 consists of amino acids 242-580 encoded by the IE2 gene of HCMV (strain AD169). Neither this late protein nor any other of the IE1/IE2-encoded proteins was detectable in extracellular virions. © 1993 Academic Press. All rights reserved.