We have analyzed human papillomavirus (HPV) type 16 RNA expression in premalignant cervical lesions of different severity and in squamous cervical cancers by RNA-RNA in situ hybridization in order to find differences in the topographic distribution of viral RNA, which might correlate with the severity of disease. In the basal layer of low-grade squamous intraepithelial lesions (SIL) only weak transcription of viral early genes was observed. Signal intensity increased strongly in the more differentiated cells accompanied by high levels of HPV DNA replication. This pattern of viral gene expression, together with the onset of viral late transcription in the upper differentiated layer of the epithelium, most likely reflects the productive phase of viral infection. In contrast, in high-grade SIL viral transcription was comparatively strong in basal cells and evenly distributed throughout the undifferentiated epithelium. This difference of viral transcription in the basal layer of the respective lesions points to an altered regulation of viral gene expression which may be causally linked to the progression of precursor lesions. Evidence for disrupted expression of 3' early genes (E2, E4, and E5), analogous to the situation in HPV-DNA containing cervical carcinoma-derived cell lines, was not found in any of the HPV-16-positive premalignant lesions nor in the majority of cancers. The similarity of the viral transcription pattern of high-grade SIL and cancers suggests that additional host gene alterations are necessary for malignant progression.