The genomic region encoding the assembly protein of simian cytomegalovirus (CMV) strain Colburn has been cloned, sequenced, and found to be organized as a nested set of four in-frame, 3'-coterminal genes, each with its own TATA promoter element and translational start codon, and all using a single 3' polyadenylation signal. The 3' end of the longest open reading frame (1.770 bp) was identical to the 930-bp sequence coding for the assembly protein precursor, as determined from a cDNA clone. The assembly protein coding region of human CMV strain AD169 was similarly organized, suggesting that both viral genomes could give rise to four independently transcribed 3'-coterminal RNAs coding for four overlapping, in-frame, carboxy-coterminal proteins. These predictions were tested and confirmed. Four mRNAs corresponding in size and sequence to those predicted were identified in both human and simian CMV-infected cells by using transcript-specific antisense oligonucleotide probes in Northern (RNA blot) assays. The 5' ends of the three largest of these Colburn transcripts were determined by S1 nuclease protection assays and found to map between the anticipated TATA sequences and corresponding translational start codons. The four predicted overlapping proteins were identified by immunoassays in lysates of simian and human CMV-infected cells by using an antiserum specific for the carboxyl end of the assembly protein precursor. The structural relationship of both sets of proteins was verified by comparing their peptide patterns following protein cleavage at tryptophan residues by N-chlorosuccinimide. The similar organization of the homologous coding regions in other herpesviruses into at least two nested, in-frame, 3'-coterminal genes is discussed.