We have examined transcription from the human cytomegalovirus immediate-early genes that are located at approximately 0.24 map units of the viral genome. Upon infection of permissive cells, nonoverlapping transcripts of 1.65 and 1.7 kb were abundant 8 h after infection and at lower levels at later times of infection or in the presence of cycloheximide. A transcript of approximately 3.4 kb that spanned the entire region was less abundant than the smaller RNAs at 8 h after infection, but was not decreased in abundance with cycloheximide treatment. These RNA species appear to correspond to the immediate-early transcripts from this region [1]. Polyadenylated RNA was isolated from human cytomegalovirus infected cells 8 h after infection and used to synthesize directly a cDNA library. cDNAs corresponding to the abundant 1.65- and 1.7-kb transcripts of this region were isolated and characterized.