Cloned sub-genomic fragments of human cytomegalovirus strain AD169 were used to analyse immediate-early (IE) transcription in virus-infected cells. Transcriptionally active regions of the HCMV genome were identified by hybridising cytoplasmic IE poly(A)+-RNA with dot blots and Southern transfers of restriction endonuclease digests of recombinant plasmids. The size, number and, in some cases, the orientation of transcription of IE RNA species were determined. The most abundant IE mRNA (IE-1.95) was mapped at 0.0764-0.0865 map units. The transcription of two middle abundant (1.7 and 2.15 kb) IE RNAs was initiated immediately downstream, and in the same orientation as the IE-1.95 gene. A second transcriptionally active area was identified at 0.593-0.619 map units. Three mRNA species (IE-1.75, IE-3.8 and IE-4.8) were derived from this region. Additional minor IE transcription was also observed from other regions of the HCMV genome. Hybrid-selected translation was used to identify the polypeptides encoded by the major IE RNA species.