The nucleotide sequence of the major immediate early (IE) gene of human cytomegalovirus strain AD169 was determined. The structure of the gene was examined by nuclease mapping and by sequence analysis of a cDNA clone made from IE mRNA. The gene encodes a spliced molecule of 1736 nucleotides, made up of four exon sequences of 121, 88, 185 and 1342 nucleotides. Three introns (827, 114 and 170n) were located near the 5' end of the gene. A single open reading frame starting in the second exon extends for 491 amino acids, corresponding to a protein of molecular weight 64000. The putative promoter region contains several short direct and inverted repeat sequences of 16, 18, 19 and 21 nucleotides, which extend 509n upstream from the transcription start site. The structure of the major IE gene and its protein product are discussed and compared with the corresponding IE gene from the Towne strain of HCMV.