Human papillomavirus type 16 (HPV16) transcription in HPV16-positive vulvar intraepithelial neoplasia (VIN), cervical intraepithelial neoplasia (CIN) and cervical carcinomas was analyzed using RNA-RNA in situ hybridization. Subgenomic probes were constructed which specifically detected individual spliced E6/E7 transcripts as well as transcripts initiated within the E7 open reading frame (ORF). In most biopsies, viral RNA was predominantly initiated in the E6 ORF at promoter P97 and contained the E6*I splice. Three of 7 VIN, 13 of 37 CIN and I of 13 cervical carcinomas expressed significant amounts of mRNA that were initiated within the E7 ORF. Promoter activity in the E7 ORF correlated with epithelial differentiation and viral late gene (LI) expression. Our data therefore do not support the finding of Böhm et al. (1993) which suggested that the predominant transcript(s) in HPV16-associated high-grade neoplasms and genital carcinomas is initiated within the E7 ORF. Rather, our data suggest that the major HPV16 transcript in high-grade cervical neoplasms and carcinomas is initiated in the E6 ORF and encodes the E7 oncoprotein.