A comprehensive analysis of 53 penile carcinomas was performed to determine which mechanisms might be involved in the disruption of the p16INK4A/cyclin D/Rb pathway. To that end, human papillomavirus (HPV) presence, p16INK4A expression and promoter methylation, and expression of the BMI-1 polycomb gene product were studied. Sixteen (30%) of the carcinomas were found to harbour high-risk HPV DNA, 15 of which contained HPV 16. HPV 16 E6/E7 oncogene transcripts were detected in 13 (87%) of the carcinomas that contained HPV 16. Strong immunostaining for p16INK4A was significantly more frequent in carcinomas that contained high-risk HPV DNA (p < 0.001) and amongst those with HPV 16 DNA, it was more frequent in lesions in which E6/E7 transcripts were detectable (p = 0.029). This supports an active role for HPV E7 in interfering with the p16INK4A/cyclin D/Rb pathway. Methylation of the p16INK4A promoter or overexpression of the BMI-1 polycomb gene product may provide alternative modes of interference with this pathway. These phenomena were mutually exclusive and found in the absence of HPV in 15% and 10% of the penile carcinomas, respectively. These data indicate that there are at least three plausible mechanisms by which the p16INK4A/cyclin D/Rb pathway can become disrupted during penile carcinogenesis. Copyright © 2003 John Wiley & Sons, Ltd.