We used a nested polymerase chain reaction (PCR) to study the presence of human papillomavirus (HPV) DNA in stained, archival cervical cytologic smears, where MY09/MY11 served as outer primers and GP5/GP6 as inner primers. It was found to give a higher positivity rate than PCR using the E1 degenerate consensus primers, where the sensitivity was decreased to 80%. It seemed optimal to use less sample DNA (0.5%) for the reaction; larger volumes resulted in decreased reactivity. Similarly, the presence of bovine serum albumin helped to improve the reactivity. The risk of cross-contamination did not seem to be a major obstacle to a valid analysis. The prevalence of HPV in normal smears was 10%, and in the high grade squamous intraepithelial lesion group it was 80%. Smears with cytologic evidence of HPV gave 100% positivity, while those containing cancer cells gave 80%. In patients whose Southern blot had demonstrated the presence of HPV, 87% of the simultaneously taken smears were also positive with nested PCR. Similarly, in those whose Southern blot analysis was negative, the corresponding smear was positive in 41%; this reactivity was associated with simultaneous squamous intraepithelial lesions. The prevalence values indicated that this analysis is both sensitive and specific and that it can be used to evaluate the performance of other diagnostic methods. The validity is sufficient to allow retrospective cohort studies of the natural history of HPV infections during carcinogenesis.