We have characterized the expression of human cytomegalovirus immediate early (IE) genes encoding the UL36-38 open reading frames. Single-stranded RNA hybridization probes, transcribed from UL36-38 cDNAs or smaller exon-specific clones, were used in analyses of RNA isolated during various phases of infection in the presence and absence of inhibitors of protein and HCMV DNA synthesis. There are three IE transcripts that arise by the use of alternative promoters and splicing signals. Different patterns of expression are observed for each of the three IE RNAs during permissive infection. Comparison of nucleotide sequences of UL36-38 IE cDNAs with those of the genomic sequence verified the use of polyadenylation and splice signals. An additional abundant early class RNA, totally encoded within the UL36-38 region, was found to be expressed from a different promoter than the IE RNAs. In vitro transcription and translation of the IE cDNAs were used to express the UL36-38 IE proteins. These studies define the structure of the UL36-38 gene products and demonstrate that expression from the region is both qualitatively and temporally complex.