Infection with genital human papillomaviruses (HPVs) is the primary cause of cervical cancer. The infection is widespread, and little is known about the secondary factors associated with progression from subclinical infection to invasive carcinoma. Here we report that HPV genomes are efficiently targeted in vivo by CpG methylation, a well-known mechanism of transcriptional repression. Indeed, it has been shown previously that in vitro-methylated HPV type 16 (HPV-16) DNA is transcriptionally repressed after transfection into cell cultures. By using a scan with the restriction enzyme McrBC, we observed a conserved profile of CpG hyperand hypomethylation throughout the HPV-16 genomes of the tumor-derived cell lines SiHa and CaSki. Methylation is particularly high in genomic segments overlying the late genes, while the long control region (LCR) and the oncogenes are unmethylated in the single HPV-16 copy in SiHa cells. In 81 patients from two different cohorts, the LCR and the E6 gene of HPV-16 DNA were found to be hypermethylated in 52% of asymptomatic smears, 21.7% of precursor lesions, and 6.1% of invasive carcinomas. This suggests that neoplastic transformation may be suppressed by CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression. These prevalences of hyper-and hypomethylation also indicate that CpG methylation plays an important role in the papillomavirus life cycle, which takes place in asymptomatic infections and precursor lesions but not in carcinomas. Bisulfite modification revealed that in most of the HPV-16 genomes of CaSki cells and of asymptomatic patients, all 11 CpG dinucleotides that overlap with the enhancer and the promoter were methylated, while in SiHa cells and cervical lesions, the same 11 or a subset of CpGs remained unmethylated. Our report introduces papillomaviruses as models to study the mechanism of CpG methylation, opens research on the importance of this mechanism during the viral life cycle, and provides a marker relevant for the etiology and diagnosis of cervical cancer.Human papillomavirus type 16 (HPV-16) and related HPV types are carcinogenic, and persistent HPV, infection is a prerequisite in the etiology of most or even all cervical cancers (22, 38, 52). Most women become infected by HPVs, and while some of these infections progress malignantly, most remain subclinical or lead only to precursor lesions. The factors that determine these outcomes are poorly understood. Transformation by HPVs depends on the oncoproteins E6 and E7, whose transcription is modulated by numerous transcription factors and epigenetic mechanisms (5, 18). Tumor progression may result from stimulated oncoprotein expression through transcriptional induction by steroids (10, 17), by deletion of transcriptional silencers (25), or by integration of HPV genomes into cellular DNA (3,40,43).cis-responsive elements that regulate E6 and E7 oncogene transcription are spread throughout the long control region (LCR) of HPV-16, an 850-bp segment between the L1 and E6 ...