Erythroplasia of Queyrat is a carcinoma in situ that mainly occurs on the glans penis, the prepuce, or the urethral meatus of elderly males. Up to 30% progress to squamous cell carcinoma. The cause of erythroplasia of Queyrat is largely unknown. Human papillomavirus type 16 DNA has previously been detected only in very few distinctly characterized patients. We have investigated 12 paraffin-embedded biopsies from eight patients with penile erythroplasia of Queyrat and control biopsies of inflammatory penile lesions, of genital Bowen's disease, and of premalignant/malignant cervical or vulvar lesions by 10 different polymerase chain reaction protocols for the presence of cutaneous and genital/mucosal human papillomaviruses. Human papillomavirus typing was performed by sequencing (cloned) polymerase chain reaction products. Human papillomavirus DNA was detected in all erythroplasia of Queyrat patients and in none of the controls with inflammatory penile lesions. The rare cutaneous carcinogenic epidermodysplasia verruciformis-associated human papillomavirus type 8 was present in all erythroplasia of Queyrat patients and the genital high-risk human papillomavirus type 16 in seven of eight patients (88%). In addition to human papillomavirus type 8 and human papillomavirus type 16, four patients carried the genital carcinogenic human papillomavirus type 39 and/or type 51. All human papillomavirus type 8 sequences found in erythroplasia of Queyrat showed some polymorphism among each other and differed in specific nucleotide exchanges from the human papillomavirus type 8 reference sequence. Viral load determinations (human papillomavirus copies/beta-globin gene copies) by realtime polymerase chain reactions showed that the human papillomavirus type 16 levels in the erythroplasia of Queyrat biopsies were one to five orders of magnitude higher than the human papillomavirus type 8 levels. Human papillomavirus type 8 was not detected in cervical or vulvar precancerous and cancerous lesions and in Bowen's disease lesions that carried genital human papillomavirus types. The data suggest that in erythroplasia of Queyrat, in contrast to other genital neoplasias, a coinfection with human papillomavirus type 8 and carcinogenic genital human papillomavirus types occurs. The presence or absence of human papillomavirus type 8 might help to distinguish between penile erythroplasia of Queyrat and Bowen's diseases.