The presence of human papillomavirus (HPV) DNA was assessed in biopsies of tonsillar carcinomas (n = 10) and cases of tonsillitis (n = 7), serving as controls, by general-primer-mediated PCR (GP-PCR). All carcinomas appeared HPV-positive, whereas all cases of tonsillitis were HPV-negative. Additional type-specific PCR for HPV 6, 11, 16, 18, 31 and 33 revealed that 4 carcinomas contained HPV 16 DNA, 4 contained HPV 33 DNA and I contained an HPV 16/33 double infection. False positivity was excluded by additional Southern blot analysis of type-specific PCR-positive samples (n = 4). Further characterization of GP-PCR products by sequence analysis revealed that 2 carcinomas contained still uncharacter-ized HPV genotypes; one of these also contained HPV 33 DNA and one was negative by type-specific PCR. Application of RNA PCR revealed expression of HPV 16 or HPV 33 E7 encoding spliced E6*l transcripts in all tonsillar carcinomas (n = 4) examined. Additional non-radioactive RNA in situ hybridization performed on 3 biopsies revealed the presence of HPV 16 or HPV 33 E7 transcripts exclusively localized within the carcinoma cells, whereas stroma stained negative. These findings strongly support a role for certain HPV types in the pathogenesis of tonsillar carcinomas.