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The DNA templates containing immediate early (IE) genes of human cytomegalovirus (CMV) were transcribed in vitro by using a HeLa cell extract. When IE region 1, 2, and 3 were used, transcription was detected qualitatively only from IE region 1. Transcription was detected with DNA representing IE region 2 when the IE region 1 promoter was not present. DNA sequence analysis of the upstream regulatory region of IE region 1 detected two distinct repeats of 19 and 18 nucleotides, both being repeated four times. A putative cruciform structure could form through the surrounding sequences with each 18-nucleotide repeat being located in the unpaired region. The potential secondary structure and the repeat sequences in the regulatory region of IE region 1 are presumably related to the high level of transcription of this IE gene.