Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex families of overlapping messenger RNAs that are linked to differentiation, making it necessary to analyze gene expression in the context of morphology. We have developed HPV type 16 and type 18 subgenomic clones from which 3H-labeled riboprobes specific to individual mRNA families can be generated in vitro. Using these probes for in situ hybridization, we examined serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade squamous lesions, all viral open reading frames were expressed, and the most abundant transcription spanned the E4 and E5 open reading frames at the 3′ end of the E region. L region transcription coding for the capsid proteins was restricted to terminally differentiated keratinocytes. As the grade of neoplasia increased, cellular differentiation and overall viral transcription decreased and, with few exceptions, the L2 and L1 transcripts ceased to exist. The E6-E7 transforming region was invariably derepressed. Interestingly, the patterns of HPV-16 gene expression suggested the coexistence of episomal and integrated viral DNAs. In contrast, in HPV-18 lesions, all the viral template DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the E1 and E2 open reading frames. Viral transcription patterns were similar in carcinomas in situ and in invasive carcinomas, regardless of the histologic cell types or the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression. On the basis of viral gene expression in vivo and the E6 promoter regulation previously characterized in vitro, we discuss a molecular mechanism for HPV-associated carcinogenesis. © 1992.